Pharmaceutical compositions comprising an adenosine receptor agonist or antagonist

ABSTRACT

Adenosine receptor agonists, particularly an agonist which binds to the A3 adenosine receptor, are used for induction of production or secretion of G-CSF within the body, prevention or treatment of toxic side effects of a drug or prevention or treatment of leukopenia, particularly drug-induced leukopenias; and inhibition of abnormal cell growth and proliferation.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/IL00/00550 which has an Internationalfiling date of Sep. 8, 2000, which designated the United States ofAmerica.

FIELD OF THE INVENTION

The present invention is generally in the field of cancer and concerns acancer therapy or a therapy intended to counter the side effect ofcancer treatment.

PRIOR ART

The following is a list of prior art which is considered to be pertinentfor describing the state of the art in the field of the invention.Acknowledgement of these references herein will be made by indicatingthe number from their list below within brackets.

-   -   1. Linden J. The FASEB J. 5:2668–2676 (1991);    -   2. Stiles G. L. Clin. Res. 38:10–18 (1990);    -   3. Stolfi R. L., et al. Cancer Res. 43:561–566 (1983);    -   4. Belardinelli L. et al. Prog. Cardiovasc. Dis. 32:73–97        (1989);    -   5. Collis M. G., Pharmacol. Ther. 41:143–162 (1989);    -   6. Clark B. and Coupe M. Int. J. Cardiol. 23:1–10 (1989);    -   7. Dubey R. K. et al. Circulation 96:2656–2666 (1997)    -   8. Soderback U. et al. Clin. Sci. 81:691–694 (1994);    -   9. Gilbertsen R. B. Agents actions 22:91–98 (1987);    -   10. Bouma M. G. et al. J. Immunol. 153: 4159–4168 (1994);    -   11. Rozengurt E. Exp. Cell Res. 139:71–78 (1982);    -   12. Gonzales F. A., et al., PNAS USA 87:9717–9721 (1990);    -   13. Sandberg G. and Fredholm B. B., Thymus 3:63–75 (1981);    -   14. Pastan I. H. et al. Annu. Rev. Biochem. 44:491–495 (1975);    -   15. WO 99/02143;    -   16. Fishman P., et al. Cancer Res. 58:3181–3187 (1998);    -   17. Djaldetti M. et al. Clin. Exp. Metastasis 14:189–196 (1996);    -   18. Fishman P. et al. Cancer Research 58:3181–3187 (1998).

BACKGROUND OF THE INVENTION

Myelotoxicity is a prevailing, severe, complication of chemotherapy andis one of the factors that limit the administrable dose of thechemotherapeutic drug. It causes more life threatening patient morbidityand actual mortality than any other chemotherapeutic side effect and mayresult in an increased number of hospital stay days. In addition, druginduced myelosuppression limits the administration of larger,potentially more effective doses of chemotherapy to patients withmalignancies. Several approaches to resolve this adverse event haveincluded the use of lithium, prostaglandin E, interferon, lactoferrinand the growth factors granulocyte-macrophage colony stimulating factor(GM-CSF) and granulocyte-colony stimulating factor (G-CSF). To date, useof growth factors such as G-CSF is a standard therapy for cancerpatients with neutropenia. It stimulates the proliferation anddifferentiation of hematopoietic progenitors and also controls thefunctional activities of neutrophils and macrophages. However, the G-CSFtreatment is costly and as it is a recombinant protein, it hasaccompanying side effects.

Adenosine, an endogenous purine nucleoside, is ubiquitous in mammaliancell types. Adenosine present in the plasma and other extracellularfluids mediates many of its physiological effects via cell surfacereceptors and is an important regulatory protein. It is released intothe extracellular environment from metabolically active or stressedcells. It is known to act through its binding to specific G-proteinassociated A1, A2 and A3 membranal receptors⁽¹⁻²⁾. The interaction ofadenosine with its receptors initiates signal transduction pathways,mainly the adenylate cyclase effector system, which utilizes cAMP as asecond messenger. While A1 and A3 receptors, which are coupled with Giproteins, inhibit adenylate cyclase and lead to a decrease in the levelof intracellular cAMP, the A2 receptor, which is coupled to Gs proteins,activates adenylate cyclase, thereby increasing cAMP levels⁽³⁾.

Since specific surface receptors for adenosine are found in nearly allcells, almost all organ systems of the body are regulated to some extentby its local release. This includes regulation of theelectrophysiological properties of the heart, sedation and suppressionof neurotransmitter's release and regulation of rennin release andvascular tone in the kidney⁽⁴⁻⁷⁾. Adenosine exerts various effects onthe immune system including anti-inflammatory activity through theinhibition of cytokine release, inhibition of platelet aggregation,induction of erythropoietin production and modulation of the lymphocytefunction⁽⁸⁻¹⁰⁾. Further, adenosine was found to play a role in themodulation of some central nervous system (CNS) functions, in woundhealing, in diuresis and in controlling pain. It was also demonstratedthat adenosine is capable of inducing proliferation in a wide range ofnormal cell types⁽¹¹⁻¹⁴⁾. This modulation of cell growth is likelymediated through the adenylate cyclase effector system described above.

In a recent study it was found that adenosine acts as a chemoprotectiveagent, which activity is likely related to its capability to stimulatebone marrow cell proliferation. Further, it was found that adenosineexerted an inhibitory effect on the proliferation of tumor cells,apparently through G0/G1 cell cycle arrest and reduction of thetelomeric signal⁽¹⁷⁻¹⁸⁾. The dual effect has turned adenosine into anattractive concept for cancer treatment.

SUMMARY OF THE INVENTION

In accordance with the present invention it was found that adenosine A3receptor agonists (A3RAg) have a dual effect in that they inhibitproliferation of malignant cells on the one hand, and counter toxic sideeffects of chemotherapeutic drugs on the other hand. Specifically, theA3RAg compounds inhibit proliferation and growth of tumor cells,synergize with an anti-tumor cytotoxic drug in reducing the tumor load,induce proliferation and differentiation of bone marrow cells and whiteblood cells and counter toxic side effects of other drugs, particularlychemotherapeutic drugs. Furthermore, it was discovered in accordancewith the invention that the A3RAg exerts these activities by a varietyof forms of administration including parenteral administration andparticularly oral administration. It was further found in accordancewith the invention that some of the A3RAg activity may be mimicked byother agonists and antagonists of the adenosine A1 or A2 receptors: theadenosine A1 receptor agonists (A1RAg) shares with the A3RAg its abilityto induce G-CSF secretion; adenosine A2 receptor agonist (A2RAg) shareswith the A3RAg its ability to inhibit proliferation of malignant cells;and the adenosine A2 receptor antagonist (A2RAn) shares with the A3RAgits ability to counter toxic side effects of drugs, e.g. treat orprevent leukopenia.

The invention relates in its broadest sense, to the use of an activeingredient to yield one of the following therapeutic/biological effects:inducing production or secretion of G-CSF within the body; prevention ortreatment of toxic side effects of a drug or prevention or treatment ofleukopenia, particularly drug-induced leukopenia; and inhibition ofabnormal cell growth and proliferation. The active ingredient may be anA3RAg or an agonist or antagonist of the adenosine receptor system whichcan yield one of these therapeutic effects, achieved by the use of theA3RAg.

Several embodiments are provided by the invention. The first embodiment,to be referred to herein as the “G-CSF-inducing embodiment” involves theuse of an active ingredient, which may be an A3RAg or an A1RAg to yieldsecretion of the G-CSF within the body of a treated subject. G-CSF isknown to stimulate proliferation and differentiation of hematopoieticprogenitors and controls the functional activities of neutrophils andmacrophages. Thus, a G-CSF-inducing agent such as those mentioned above,may have a high therapeutic value, for example, in countering (i.e.preventing, reducing, or ameliorating) myelotoxicity.

Provided in accordance with this embodiment is a method for inducingG-CSF secretion within the body of a subject, comprising administeringto the subject an effective amount of an active ingredient selected fromthe group consisting of A3RAg, an A1RAg and a combination of an A3RAgand an A1RAg. In accordance with this embodiment there is furtherprovided a method for the therapeutic treatment, comprisingadministering to a subject in need an effective amount of said activeingredient for achieving a therapeutic effect, the therapeutic effectcomprises induction of G-CSF production or secretion. Still furtherprovided by this embodiment is use of said active ingredient for themanufacture of a pharmaceutical composition for inducing G-CSFsecretion. Also provided by this embodiment is a pharmaceuticalcomposition for inducing production or secretion of G-CSF within thebody, comprising a pharmaceutically acceptable carried an effectiveamount of said active ingredient.

In accordance with another embodiment of the invention, to be referredto herein at times as the “Leukopenia-prevention embodiment” or morespecifically as the “neutropenia-prevention embodiment”, an activeingredient which may be an A3RAg, or an A2RAn, is used for theprevention or treatment of leukopenia, which may result frommyelotoxicity.

In accordance with this embodiment there is provided a method forinducing proliferation or differentiation of bone marrow or white bloodcells in a subject, comprising administering to the subject an effectiveamount of an active ingredient selected from the group consisting of anA3RAg, an adenosine A2RAn and a combination of an A3RAg or an A2RAn.Also provided by this embodiment is a method for prevention or treatmentof leukopenia, comprising administering to a subject in need aneffective amount of said active ingredient. Further provided inaccordance with this embodiment is use of said active ingredient for themanufacture of a pharmaceutical composition for inducing proliferationor differentiation of bone marrow or white blood cells. Still furtherprovided in accordance with this embodiment is use of said activeingredient for the manufacture of a pharmaceutical composition for theprevention or treatment of leukopenia. The pharmaceutical compositioncan particularly be used for prevention or treatment of leukopenia.

In accordance with a related embodiment, to be referred to herein as the“toxicity-preventing embodiment” the abovementioned active ingredient(namely one of the A3RAg, or A2RAn, as well as a combination thereof, isused to counter toxic side effects of drugs, such as chemotherapeuticdrugs or nemoleptic drugs.

In accordance with this latter embodiment there is thus provided amethod for prevention or treatment of toxic side effects of a drug,comprising administering to a subject in need an effective amount of anactive ingredient selected from the group consisting of an A3RAg, anA2RAn and a combination of an A3RAg and an A2RAn. Also provided inaccordance with this embodiment is use of said active ingredient for themanufacture of a pharmaceutical composition for the prevention ortreatment of drug-induced toxicity. Still further provided by thisembodiment is pharmaceutical composition for prevention or treatment oftoxic side effects of a drug, comprising an effective amount of saidactive ingredient and a pharmaceutically acceptable carrier.

For the purpose of countering drug-induced leukopenia or drug-inducedtoxic side effects in general, it is at times desirable to formulate adrug which has such toxic side effects together with said activeingredient for combined administration of the two. The invention thusalso provides a pharmaceutical composition comprising, in combination adrug that can cause toxic side effect in a subject treated thereby andsaid active ingredient; as well as use of said active ingredient for themanufacture of such a pharmaceutical composition. Said activeingredients included in said composition being an amount effective forprevention or treatment of the toxic side effects.

In accordance with yet another embodiment of the invention, to bereferred to herein as the “proliferation-inhibiting embodiment”, anactive ingredient, which may be an A3RAg, an A2RAg, or a combination ofthe two, is used for selectively inhibiting abnormal cell growth, e.g.tumor cell growth.

In accordance with this embodiment there is provided a method forinhibiting abnormal cell growth in a subject, comprising administeringto the subject a therapeutically effective amount of an activeingredient selected from the group consisting of an A3RAg, an A2RAg anda combination of an A3RAg and an A2RAg. Also provided in accordance withthis embodiment is use of said active ingredient for the manufacture ofa pharmaceutical composition for inhibiting abnormal cell growth. Stillfurther provided by this embodiment is a pharmaceutical composition forinhibiting abnormal cell growth, comprising said active ingredient, anda pharmaceutically acceptable carrier.

In one embodiment of the invention the administration of the activeingredient is intended to achieve dual therapeutic effect: inhibition ofabnormal cell growth and reduction of toxic side effects of a drugcausing such effects.

The preferred active ingredient in accordance with the invention is anA3RAg. The preferred route of administration of the active ingredient,in accordance with the invention is the oral administration route.However, this preference does not exclude other active ingredientsneither other administration routes of the active ingredients.

The dosage of the active ingredient, particularly where the activeingredient is an A3Rag, is preferably less than 100 μg/kg body weight,typically less than 50 μg and desirably within the range of 1–10 μg/kgbody weight.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the invention novel therapeutic use is provided forcertain active agents, particularly adenosine receptor agonists andantagonists. By one embodiment, the G-CSF-inducing embodiment, some suchagents are used to mediate the production and secretion of G-CSF fromcells. In accordance with another embodiment, the toxicity-preventingembodiment, some such agents are used to counter toxic side effects ofdrugs, e.g. chemotherapeutic or nemoleptic drugs. In a furtherembodiment, the leukopenia-prevention embodiment, some such agents areused to counter leukopenia, particularly drug-induced leukopenia. Inaccordance with yet another embodiment, the proliferation-inhibitionembodiment, some such agents are used to selectively inhibit abnormalcell growth.

The term “leukopenia” as used herein refers to the reduction in thecirculating white blood cell count. While leukopenia is usuallycharacterized by a reduced number of blood neutrophils (neutropenia), attimes, a reduced number of lymphocytes, monocytes, eosinophils orbasophils may be detected.

Leukopenia which may arise from the reduced production or excessivesplenic sequestration of neutrophils, may result from a hereditary andcongenital diseases. However it is mainly observed after treatment withdrugs, such as cytoreductive cancer drugs, antithyroid drugs,phenothiazines, anticonvulsants penicillins, sulfonamides, andchloramphenicol. Some antineoplastics cause leukopenia as a predictableside effect.

In the following, a reduction in leukocyte count or neutrophil count bydrugs will be referred to herein, as “drug-induced leukopenia” or“drug-induced neutropenia”. Furthermore, whenever mention is made toleukopenia, it should be understood as referring particularly to“neutropenia”.

Further, the term “prevention or treatment of leukopenia” should beunderstood as a procedure whereby the reduction in leukocyte cell countwhich may otherwise occur, is reduced, totally prevented or if suchreduction has occurred, a procedure which gives rise to increase in theleukocyte cell count. Leukopenia is manifested by a variety of sideeffects such as an increased possibility to infection by significantinfectious agents and others. The term “prevention or treatment ofleukopenia” should also be understood as meaning an improvement in suchparameters which may occur as a result of leukopenia.

The pharmaceutically or therapeutically “effective amount” for purposesherein is determined by such considerations as may be known in the art.The amount must be effective to achieve the desired therapeutic effectwhich depends on the type and mode of treatment. As is clear to theartisan, the amount should be effective to obtain the improvement ofsurvival rate, to obtain a more rapid recovery to obtain the improvementor elimination of symptoms or any other indicators as are selected asappropriate measures by those skilled in the art. When, for example,said active ingredient is administered to induce G-CSF production, aneffective amount of the active ingredient may be an amount which leadsto production and secretion of G-CSF from peripheral blood mononuclearcells, endothelial cell or fibroblast, in which it was produced,thereby, for example, stimulating the maturation of granulocytesprogenitors into mature neutrophils. Where the active ingredient isadministered to counter drug-induced leukopenia, an effective amount ofthe active ingredient may be an amount which protects the individualagainst the drug-induced reduction in the count of leukocytes,particularly neutrophils; an amount of the active ingredient which cangive rise to an increase in an already decreased level of such cells,e.g. restore the level to a normal level or sometimes even above; etc.Where the active ingredient is administered in order to reduce toxicside effect of a drug, the amount of the active ingredient may, forexample, be an amount effective in reduction of weight loss resultingfrom the drug administered. Where the active ingredient is administeredin order to inhibit abnormal cell growth, as detailed hereinafter, aneffective amount may be an amount which will inhibit the proliferationof such cells in the treated subject and even eliminate the tumor. Wherethe active ingredient is administered in order to potentiate the effectof an anti-cancer chemotherapeutic drug, an effective amount may be anamount which either increases the cancer specific toxicity of thechemotherapeutic treatment; an amount which is effective in reducing theamount of the chemotherapeutic drug or drug combination required toachieve a desired effect of the chemotherapeutic drug or drugcombination, i.e. reduction of the tumor load; etc. An example of aneffective amount is a daily administration of A3RAg less than 100 μg/kgbody weight, typically less than 50 μg/kg body weight and optionallyeven less than 10 μg/kg body weight, e.g. about 3–6 μg/kg body weight.Such an amount of A3RAg is typically administered in a single daily dosealthough at times a daily dose may be divided into several dosesadministered throughout the day or at times several daily doses may becombined into a single dose to be given to the patient once everyseveral days, particularly if administered in a sustained releaseformulation.

The active ingredient according to the invention is preferably an A3RAg.The A3RAg is any agonist which binds to A3 receptors and activates themto yield a therapeutic effect of the present invention. It should benoted that at times, an A3RAg may also interact with other receptors,e.g. with the A1 and A2 receptors. However, the A3RAg used in accordancewith the invention exerts its prime effect through the A3 receptor(namely there may also be minor effects exerted through interaction withother adenosine receptors).

By one embodiment, the active ingredient according to the invention is anucleoside derivative. By the term “nucleoside” it is meant any compoundcomprising a sugar, preferably ribose or deoxyribose, or a purine orpyrimidine base or a combination of a sugar with a purine or pyrimidinebase preferably by way of N-glycosyl link. The term“nucleoside-derivative” will be used to denote herein a naturallyoccurring nucleoside as defined hereinabove, a synthetic nucleoside or anucleoside which underwent chemical modifications by way of insertion/s,deletion/s or exocyclic and endocyclic substitution/s of group/s thereinor conformational modifications which provide a derivative with thedesired biological effect.

In accordance with one preferred embodiment of the invention the activeingredient is an A3RAg.

According to one embodiment of the invention, the active ingredient is anucleoside derivative of the following general formula (I):

-   -   wherein R₁ is C₁–C₁₀ alkyl, C₁–C₁₀ hydroxyalkyl, C₁–C₁₀        carboxyalkyl or C₁–C₁₀ cyanoalkyl or a group of the following        general formula (II):

-   -   in which:        -   Y is an oxygen or sulfur atom or CH₂;        -   X₁ is H, C₁–C₁₀ alky, R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein            R^(a) and R^(b) may be the same or different and are            selected from the group consisting of hydrogen, C₁–C₁₀            alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀            BOC-aminoalkyl, and C₃–C₁₀ cycloalkyl or are joined together            to form a heterocyclic ring containing two to five carbon            atoms, and R^(c) is selected from the group consisting of            C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl,            C₁–C₁₀ BOC-aminoalkyl, and C₃–C₁₀ cycloalkyl;        -   X₂ is H, hydroxyl, C₁–C₁₀ alkylamino, C₁–C₁₀ alkylamido or            C₁–C₁₀ hydroxyalkyl;        -   X₃ and X₄ each independently are hydrogen, hydroxyl, amino,            amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro,            trifluoro, aryl, alkaryl, thio, thioester, thioether,            —OCOPh, —OC(═S)OPh or both X₃ and X₄ are oxygen connected            to >C═S to form a 5-membered ring, or X₂ and X₃ form the            ring of formula (III):

-   -   where R′ and R″ are independently C₁–C₁₀ alkyl;        -   R₂ is selected from the group consisting of hydrogen, halo,            C₁–C₁₀ alkylether, amino, hydrazido, c₁–C₁₀ alkylamino,            C₁–C₁₀ alkoxy, C₁–C₁₀ thioalkoxy, pyridylthio, C₂–C₁₀            alkenyl, C₂–C₁₀ alkynyl, thio, and C₁–C₁₀ alkylthio; and        -   R₃ is an —NR₄R₅ group, with R₄ being hydrogen, alkyl,            substituted alkyl or aryl-NH—C(Z)—, with Z being O, S or            NR^(a) with R^(a) having the above meanings, and, when        -   R₄ is hydrogen, R₅ being selected from the group consisting            of R- and S-1-phenylethyl, benzyl, phenylethyl or anilide            groups, each such group being unsubstituted or substituted            in one or more positions with a substituent selected from            the group consisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀            haloalkyl, nitro, hydroxyl, acetamido, C₁–C₁₀ alkoxy, and            sulfonic acid or a salt thereof; or R₅ being            benzodioxanemethyl, fururyl, L-propylalanylaminobenzyl,            β-alanylaminebenzyl, T-BOC-β-alanylaminobenzyl, phenylamino,            carbamoyl, phenoxy or C₁–C₁₀ cycloalkyl; or R₅ being a group            of the following formula:

or, when R₄ is, alkyl, substituted alkyl, or aryl-NH—C(Z)—, then R₅being selected from the group consisting of substituted or unsubstitutedheteroaryl-NR^(a)—C(Z)—, heteroaryl-C(Z)—, alkaryl-NR^(a)—C(Z)—,alkaryl-C(Z)—, aryl-NR-C(Z)— and aryl-C(Z)—, with Z having the abovedefined meanings;

-   -   or a suitable salt of the compound defined above, e.g. a        triethylammonium salt thereof.

According to this embodiment of the invention, the active ingredient ispreferably a nucleoside derivative of the general formula (IV):

-   -   wherein X₁, R₂ and R₄ are as defined above.

Preferred active ingredients according to this embodiment of theinvention may generally be referred to asN⁶-benzyladenosine-5′-uronamides and derivatives thereof found to beA3-selective adenosine receptor agonists. Examples for such derivativesare N⁶-2-(4-aminophenyl)ethyladenosine (APNEA),N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide) (AB-MECA) and1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl)-N-methyl-β-D-ribofuranuronamide,the latter also referred to in the art asN⁶-3-iodobenzyl-5′-methylcarboxamidoadenosine,N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronimide and herein above andbelow by the abbreviation IB-MECA or a chlorinated derivative of IB-MECA(R₂=Cl), referred to herein as Cl-IB-MECA, IB-MECA and Cl-IB-MECA beingcurrently particularly preferred.

According to another embodiment of the invention, the active ingredientmay be an adenosine derivative generally referred to asN⁶-benzyladenosine-5′-N-alkyluronamide-N¹-oxide orN⁶-benzyladenosine-5′-N-dialyluronamide-N¹-oxide.

Yet further, the active ingredient may be a xanthine-7-ribosidederivative of the following general formula (V):

-   -   wherein:        -   X is or S;        -   R₆ is R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein        -   R^(a) and R^(b) may be the same or different and are            selected from the group consisting of hydrogen, C₁–C₁₀            alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, and            C₃–C₁₀ cycloalkyl, or are joined together to form a            heterocyclic ring containing two to five carbon atoms; and        -   R^(c) is selected from C₁–C₁₀ alkyl, amino, C₁–C₁₀            haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀ BOC-aminoalkyl and            C₃–C₁₀ cycloalkyl;        -   R₇ and R₈ may be the same or different and are selected from            the group consisting of C₁–C₁₀ alkyl, C₁–C₁₀ cycloalkyl, R-            or S-1-phenylethyl, an unsubstituted benzyl or anilide            group, and a phenylethyl or benzyl group substituted in one            or more positions with a substituent selected from the group            consisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl,            nitro, hydroxyl, acetamido, C₁–C₁₀ alkoxy, and sulfonic            acid;        -   R₉ is selected from the group consisting of halo, benzyl,            phenyl, C₃–C₁₀ cylcycloalkyl, and C₁–C₁₀ alkoxy; or a salt            of such a compound, for example, a triethylammonium salt            thereof.

Some of the above defined compounds and their synthesis procedure may befound in detail in U.S. Pat. No. 5,688,774; U.S. Pat. No. 5,773,423,U.S. Pat. No. 6,048,865, WO 95/02604, WO 99/20284 and WO 99/06053,incorporated herein by reference.

More specifically, the following specific examples are specified in U.S.Pat. No. 5,688,774 at column 4, line 67; column 5, line 16; column 5,lines 39–45; column 6, lines 21–42; column 7, lines 1–11; column 7,lines 34–36; and column 7, lines 60–61:

-   N⁶ (3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-hydroxyethyladenine;-   R—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl) adenine;-   S—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl) adenine;-   N⁶-(3-iodobenzyladenin-9-yl)acetic acid;-   N⁶-(3-iodobenzyl)-9-(3-cyanopropyl)adenine;-   2-chloro-N⁶-(3-iodobenzyl)-9-methyladenine;-   2-amino-N-(3-iodobenzyl)-9-methyladenine;-   2-hydrazido-N⁶-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-2-methylamino-9-methyladenine;-   2-dimethylamino-N-6-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-methyl-2-propylaminoadenine;-   2-hexylamino-N-6-(3-iodobenzyl)-9-methyladenine;-   N⁶-(3-iodobenzyl)-2-methoxy-9-methyladenine;-   N⁶-(3-iodobenzyl)-9-methyl-2-methylthioadenine;-   N⁶-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine;-   (1S,2R,3S,4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2,3-triol;-   (1S,2R,3S,4R)-4-(6-amino-2-chloro-9H-purin-9-yl)    cyclopentane-1,2,3-triol;-   (±)-9-[2α,    3α-dihydroxy-4β-(N-methylcarbamoyl)cyclopent-1β-yl)]-N⁶-(3-iodobenzyl)-adenine;-   2-chloro-9-(2′-amino-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(2′,3′-dideoxy-2′-fluoro-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl    adenine;-   9-(2-acetyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(3-deoxy-2-methanesulfonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(3-deoxy-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-β-D-5-ribofuranosyl)-N⁶-(3-iodobenzyl)adenine;-   2-chloro-9-(2′,3′-O-thiocarbonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)    adenine;-   9-(2-phenoxythiocarbonyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;-   1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-β-D-ribofuranosiduronamide;-   2-chloro-9-(2,3-dideoxy-β-D-5-methyl-ribofuronamido)-N⁶-benzyladenine;-   2-chloro-9-(2′-azido-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-benzyladenne;-   2-chloro-9-(β-D-erythrofuranoside)-N⁶-(3- iodobenzyl)adenine;-   N⁶-(benzodioxanemethyl)adenosine;-   1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-62-D-ribofuranosiduronamide;-   N⁶-[3-(L-prolylamino)benzyl]adenosine-5′-N-methyluronamide;-   N⁶-[3-(β-alanylamino)benzyl]adenosine-5′-N-methyluronamide;-   N⁶-[3-(N—T-Boc-β-alanylamino)benzyl]adenosine-5′-N-methyluronamide-   6-(N′-phenylhydrazinyl)purine-9-β-ribofuranoside-5′-N-methyluronamide;-   6-(O-phenylhydroxylamino)purine-9-β-ribofuranoside-5′-N-methyluronamide;-   9-(β-D-2′,3′-dideoxyerythrofuranosyl)-N⁶-[(3-β-(alanylamino)benzyl]adenosine;-   9-(β-D-erythrofuranoside)-2-methylamino-N⁶-(3-iodobenzyl)adenine;-   2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine;-   2-chloro-(2′-deoxy-6′-thio-L-arabinosyl)adenine; and-   2-chloro-(6′-thio-L-arabinosyl)adenine.

In U.S. Pat. No. 5,773,423 at column 6, line 39, to column 7, line 14,preferred compounds include those of the formula:

wherein X₁ is R^(a)R^(b)NC (═O), wherein R^(a) and R^(b) may be the sameor different and are selected from the group consisting of hydrogen,C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, and C₃–C₁₀cycloalkyl, R₂ is selected from the group consisting of hydrogen, halo,C₁–C₁₀ alkyoxy, amino, C₂–C₁₀ alkenyl, and C₂–C₁₀ alkynyl, and R₅ isselected from the group consisting of R- and S-1-phenylethyl, anunsubstituted benzyl group, and a benzyl group substituted in one ormore positions with a substituent selected from the group consisting ofC₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl, nitro, hydroxy, acetamido,C₁–C₁₀ alkoxy, and sulfo. More preferred compounds include those of theabove formula wherein R^(a) and R^(b) may be the same or different andare selected from the group consisting of hydrogen and C₁–C₁₀ alkyl,particularly when R₂ is hydrogen or halo, especially hydrogen.Additional preferred compounds are those compounds wherein R^(a) ishydrogen and R₂ is hydrogen, particularly when R₅ is unsubstitutedbenzyl. More preferred compounds are such compounds wherein R^(b) is aC₁–C₁₀ alkyl or C₃–c₁₀ cycloalkyl, particularly a C₁–C₁₀ alkyl, and moreparticularly methyl. Especially preferred are those compounds whereR^(a) is hydrogen, R^(b) is C₁–C₁₀ alkyl or C₃–C₁₀ cycloalkyl, and R₅ isR- or S-1-phenylethyl or a benzyl substituted in one or more positionswith a substituent selected from the group consisting of halo, amino,acetamido, C₁–C₁₀ haloalkyl, and sulfo, where the sulfo derivative is asalt, such as a triethylammonium salt. An example of an especiallypreferred compound is IB-MECA. In addition, those compounds in which R₂is a C₂–C₁₀ alkyne of the formula R^(d)—C═C— where R^(d) is a C₁–C₈alkyl are particularly preferred. Also preferred are those compoundswherein R₂ is other than hydrogen, particularly those wherein R₂ ishalo, C₁–C₁₀ alkylamino, or C₁–C₁₀ alkylthio, and, more preferably, whenadditionally R^(a) is hydrogen, R^(b) is a C₁–C₁₀ alkyl, and/or R₅ is asubstituted benzyl. Such preferred compounds include2-chloro-N⁶-(3-iodobenzyl)-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine,N⁶-(3-iodobenzyl)-2-methylamino-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine,andN⁶-(3-iodobenzyl)-2-methylthio-9-[5-(methylamido)-β-D-ribofuranosyl]-adenine.

Additional preferred compounds are specified in U.S. Pat. No. 5,773,423at column 7, line 60, through column 8, line 6, as modifiedxanthine-7-ribosides having the formula:

Particularly preferred are those compounds wherein X is O, R₆ isR^(a)R^(b)NC(=), wherein R^(a) and R^(b) may be the same or differentand are selected from the group consisting of hydrogen, C₁–C₁₀ alkyl,amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, and C₃–C₁₀ cycloalkyl, R₇and R₈ may be the same or different and are selected from the groupconsisting of C₁–C₁₀ alkyl, R- and S-1- phenylethyl, an unsubstitutedbenzyl group, and a benzyl group substituted in one or more positionswith a substituent selected from the group consisting of C₁–C₁₀ alkyl,amino, halo, C₁–C₁₀ haloalkyl, nitro, hydroxy, acetamido, C₁–C₁₀ alkoxy,and sulfo, and R₉ is selected from the group consisting of halo, benzyl,phenyl, and C₃–C₁₀ cycloalkyl.

WO 99/06053 discloses in examples 19–33 and originally filed claim 13,compounds selected from the group consisting of:

-   N⁶-(4-biphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(4-methoxyphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(4-chlorophenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(phenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(benzylcarbamoylamino)-adenosine-5′-N-ethyluronamide;-   N⁶-(4-sulfonamido-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-(4-acetyl-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-((R)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-((S)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-(4-n-propoxy-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;-   N⁶-bis-(4-nitrophenylcarbamoyl)-adenosine-5′-N-ethyluronamide; and-   N⁶-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide.

The active ingredient in the case of the GSF-inducing embodiment mayalso be an A1RAg. It is typically an adenosine derivative having thefollowing formula

-   -   R₁ represents a lower alkyl, cycloalkyl, preferably C₃–C₈        cycloalkyl (including the well known cyclohexyl and cyclopentyl        containing derivatives, recognized as CPA and CHA,        respectively), the cycloalkyl group may be substituted with, for        example, a hydroxyl or lower alkyl; R₁ also represents a        hydroxyl or hydroxyalkyl; a phenyl, anilide, or lower alkyl        phenyl, all optionally substituted by one or more substituents,        for example, halogen, lower alkyl, haloalkyl such as        trifluoromethyl, nitro, cyano, —(CH₂)_(m)CO₂R^(a),        —(CH₂)_(m)CONR₂R^(a)R^(b), —(CH₂)_(m)COR^(a), m representing an        integer from 0 to 6; —SOR^(c), —SO₂R^(c), —SO₃H,        —SO₂NR^(a)R^(b), —OR^(a), —SR^(a), —NHSO₂R^(c), —NHCOR^(a),        —NR^(a)R^(b) or NHR^(a)CO₂R^(b); wherein    -   R^(a) and R^(b) represent independently a hydrogen, lower alkyl,        alkanoyl, phenyl or naphthyl (the latter may be partially        saturated) the alkyl group optionally being substituted with a        substituted or unsubstituted phenyl or phenoxy group; or when R₁        represents —NR^(a)R^(b), said R^(a) and R^(b) form together with        the nitrogen atom a 5- or 6-membered heterocyclic ring        optionally containing a second heteroatom selected from oxygen        or nitrogen, which second nitrogen heteroatom may optionally be        further substituted by hydrogen or lower alkyl; or —NR^(a)B^(b)        is a group of general formulae (VII) or (VIII):

-   -   wherein n is an integer from 1 to 4;    -   Z is hydrogen, lower alkyl or hydroxyl;    -   Y is hydrogen, lower allyl, or OR′ where R′ is hydrogen, lower        alkyl or lower alkanoyl;    -   A is a bond or a lower alkylene, preferably, C₁–C₄ alkenyl; and    -   X and X′ are each independently hydrogen, lower alkyl, lower        alkoxy, hydroxy, lower alkanoyl, nitro, haloalkyl such as        trifluoromethyl, halogen, amino, mono- or di-lower alkyl amino,        or when X and X′ are taken together a methylenedioxy group;    -   R^(c) represents a lower allyl;    -   R₂ represents a hydrogen; halogen; substituted or unsubstituted        lower alkyl or alkenyl group; lower haloalkyl or haloalkenyl;        cyano; acetamido; lower alkoxy; lower alkylamino; NR^(d)R^(e)        where R^(d) and R^(e) are independently hydrogen, lower alkyl,        phenyl or phenyl substituted by lower alkyl, lower alkoxy,        halogen or haloalkyl such as trifluoromethyl or alkoxyl; or        —SR^(f) where R^(f) is hydrogen, lower alkyl, lower alkanoyl,        benzoyl or phenyl;    -   W represents the group —OCH₂—, —NHCH₂—, —SCH₂— or —NH(C═O)—;    -   R₃, R₄ and R₅ represent independently a hydrogen, lower alkyl or        lower alkenyl, branched or unbranched C₁–C₁₂ alkanoyl, benzoyl        or benzoyl substituted by lower alkyl, lower alkoxy, halogen, or        R₄ and R₅ form together a five membered ring optionally        substituted by a lower alkyl or alkenyl; R₃ further represents        independently a phosphate, hydrogen or dihydrogen phosphate, or        an alkali metal or ammonium or dialkali or diammonium said        thereof;    -   R₆ represents a hydrogen, halogen atom; or    -   one of the R groups (i.e. R₁ to R₆) is a sulfohydrocarbon        radical of the formula R^(g)—SO₃—R^(h)—, wherein R^(g)        represents a group selected from C₁–C₁₀ aliphatic, phenyl and        lower allyl substituted aromatic group which may be substituted        or unsubstituted and R^(h) represents a monovalent cation.        Suitable monovalent cations include lithium, sodium, potassium,        ammonium or trialkyl ammonium, which will enable dissociation to        take place under physiological conditions. The remaining R        groups being a hydrogen or halogen atom, an unsubstituted        hydrocarbon or any other non-sulfur containing group as defined        above.

The hydrocarbon chains used herein may include straight or branchedchains. In particular, the terms “alkyl” or “alkenyl” as used hereinmean a straight or branched chain alkyl or alkenyl groups. The terms“lower alkyl” or “lower alkenyl” mean respectively C₁–C₁₀ alkyl orC₂–C₁₀ alkenyl groups and preferably, C₁–C₆ alkyl and C₂–C₆ alkenylgroups.

Preferred adenosine derivatives of formula (VI) are the N⁶-cyclopentyladenosine (CPA), 2-chloro-CPA (CCPA), and N⁶-cyclohexyl adenosine (CHA)derivatives, the preparation of which is well known to the personskilled in the art. Other adenosine derivatives which are known to beselective to the A1 receptor are those wherein R₁ is a anilide group,the latter may be unsubstituted or substituted for example withhydroxyl, alkyl, alkoxy or with a group —CH₂C(O)R″, R″ being an hydroxylgroup, —NHCH₃, —NHCH₂CO₂C₂H₅,(ethyl glycinate), tuloidide (also in whichthe methyl moiety is replaced with a haloalkyl moiety), or with a group—CH₂C(O)NHC₆H₄CH₂C(O)R′″, in which R′″ represents a group to yield amethyl ester substituent (—OCH₃), an amide substituent (e.g. R′″ being agroup —NHCH₃), or R′″ being a hydrazide, ethylenediamine,—NHC₂H₅NHC(O)CH₃, 4-(hydroxy-phenyl)propionyl, biotinylated ethylenediamine or any other suitable hydrocarbon which renders the compound anA1 agonist.

Alternatively, the N⁶-substituted adenosine derivatives used as activeingredients according to the present invention may be those containingan epoxide moiety and more particularly are a cycloalkyl epoxycontaining adenosine derivative (e.g. oxabicyclo such as norbornanyl oroxatricyclo such as adamantanyl). Some such compounds may be defined bygeneral formula (I),

-   -   wherein R₁ is a group of general formulae (IXa) and (IXb):

-   -   wherein M is a lower alkyl group as defined above.

Embodiments of the agonist compounds having an epoxide N⁶-norbornylgroup include the endo and exo isomers and more particularly, can be oneof four isomers: the 2R-exo, 2R-endo, 2S-exo and 2S-endo form.

Another embodiment of the N⁶-norbornyl derivative may include an oxygenatom at the N¹-position of the purine ring. This compound is termedN⁶-(5,6-epoxynorborn-2-yl)adenosine-1-oxide.

At times, the A1RAg may be an adenine derivative in which theβ-D-ribofuranozyl moiety of adenosine is replaced with a hydrogen orphenyl group.

A2RAn, which may be used in accordance with the invention are 8-styrylderivatives of 1,3,7-substituted xanthines of the formula (X):

wherein

-   -   R₁ and R₃ are C₁–C₄ alkyl, allyl or propargyl    -   R₇ is H, methyl or C₂–C₈ alkyl    -   n is 1 to 3    -   and X is a halogen, trifluoroalkyl, alkoxy, hydroxy, nitro,        amino, dialkylamino, diazonium, isothiocyanate, benzyloxy,        aminoalkoxy, alkoxycarbonylamino, acetoxy, acetylamino,        succinylamino,        4-(4-NH₂-trans-CH₂CH═CHCH₂0-3,5-(MeO)₂,4-(4-AcNH-trans-CH₂CH═CHCH₂O)-3,5-(MeO)₂,        4-(4-t-BOC-NH-trans-CH₂CH═CHCH₂O)-3,5-(MeO)₂

A specific example of the compound of formula (X) is(3,7-dimethyl-1-propargyl-xantane).

The A2RAn may also be compounds of the following formulae:

Or,

As will be appreciated, the invention may not be limited to the specificA3RAg, A2RAg or A2RAn compounds mentioned above.

The active ingredient in accordance with the invention may be as definedabove or may be in the form of salts or solvates thereof, in particularphysiologically acceptable salts and solvates thereof. Further, whencontaining one or more asymmetric carbon atoms, the active ingredientmay include isomers and diastereoisomers of the above active ingredientsor mixtures thereof.

Pharmaceutically acceptable salts of the above active ingredientsinclude those derived from pharmaceutically acceptable inorganic andorganic acids. Examples of suitable acids include hydrochloric,hydrobromic, sulphoric, nitric, perchloric, fumaric, maleic, phosphoric,glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric,acetic, citric, methanesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic and benzenesulfonic acids.

The active ingredient may be administered as a non-active substance(e.g. pro-drug) and be made active only upon further modification/s by anatural process at a specific site in the subject. In any case, thederivative will be such that the therapeutic functionality of thepharmaceutical composition of the invention, is preserved. Suchpro-drugs are also encompassed by the term “active ingredient” as usedherein. Similarly, the terms “A3RAg”, “A1RAg” “A1RAn” “A2RAg” and“A2RAn” should be understood as encompassing pro-drugs which, although apriori, lack the antagonistic or antagonistic activity (as the case maybe), become active in vivo.

The A3RAg in accordance with the invention may be chosen by screeningfor such compounds which qualitatively have an activity resembling thatof IB-MECA. For example, such compounds for use in accordance with theleukopenia-inhibiting embodiment may be screened based on their abilityto stimulate proliferation of bone marrow or white blood cells andsubsequently based on their ability to exert this activity in vivo. Foruse in the proliferation-inhibition embodiment, compounds may bescreened for their ability to inhibit proliferation of tumor cells aswell as subsequently to exert this activity in vivo.

The A1RAn and A2RAn may be tested for their activity and screened foruse in therapy in a similar manner, mutatis mutandis, to that describedfor A3RAg.

The pharmaceutical composition of the invention may comprise the activeingredient as such, but may be combined with other ingredients which maybe a pharmaceutically acceptable carrier, diluent, excipient, additiveand/or adjuvant, as known to the artisan, e.g., for the purposes ofadding flavors, colors, lubrication or the like to the pharmaceuticalcomposition. Evidently, the pharmaceutically acceptable carrier/s,diluent/s, excipient/s, additive/s employed according to the inventiongenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating materials which preferably do not react with the compoundswithin the composition of the invention.

Further, the active ingredient may also be administered in combinationwith a chemotherapeutic drug, particularly in the case of the leukopeniaprevention embodiment. Thus the pharmaceutical composition according tothe invention may comprise, in addition to said active ingredient achemotherapeutic drug. According to one embodiment of the invention, thechemotherapeutic drug is an anti-cancer chemotherapeutic drug. It shouldbe understood that by the term it is meant any cytotoxic drug or acocktail comprising a combination of two or more cytotoxic drugs givento a patient for the purpose of reducing the patient's tumor mass.

One finding in accordance with the invention is that the A3RAg is orallybioavailable and exerts its dual activity (reducing abnormal cellproliferation and preventing or reducing leukopenia) when orallyadministered. Thus, according to one preferred embodiment, thepharmaceutical composition of the invention is formulated for oraladministration. Such an oral composition may further comprise apharmaceutically acceptable carrier, diluent, excipient, additive oradjuvant suitable for oral administration.

Within the scope of the G-CSF-inducing embodiment of the presentinvention, the pharmaceutical compositions disclosed are particularlyused for increasing the level of G-CSF secreted from the cells. Suchcompositions may be used to accelerate the neutrophil recovery afterchemotherapy and bone marrow transplantation or to inhibit abnormal cellgrowth. To date, such treatments include administration of the growthfactor it self, which are known to have undesired side effects. All themore so, the average cost per course of G-CSF therapy is known to bevery high.

Within the scope of the leukopenia-prevention embodiment or thetoxicity-preventing embodiment of the present invention, thepharmaceutical composition disclosed are particularly used for elevatingthe level of circulating leukocyte cells in a subject or counteringother toxic effects, such as weight loss. This aspect of the inventionis applicable in a variety of clinical situations. It is evident that areduced level of circulating leukocytes and particularly neutrophils mayresult in a weakened immune system. An example of a weakened immunesystem which may be treated in accordance with this aspect of theinvention, is such which often occurs in advanced stages of cancer orthat resulting from drug-induced leukopenia or drug-induced neutropenia.

The proliferation-inhibiting embodiment is useful for the treatment of avariety of abnormalities associated with the abnormal cell growth suchas cancer, psoriasis and some autoimmune diseases. In particular, thecomposition of the invention is employed for inhibiting proliferation oftumor cells, preferably within the framework of anti-cancer therapy.

When treating lymphoma cells with an A3RAg the inhibition ofproliferation of these cells was more pronounced than that obtained withadenosine or the ‘A1’ or ‘A2’ agonists, although some activity was alsoobserved with the A2RAg (see for example FIG. 5A). These results showthat inhibition of tumor cell proliferation should be ascribed mainly tothe binding of A3RAg to its corresponding receptor but may also bemimicked to some extent by an A2RAg. The above surprising results thusoffers a new therapeutic target for future anti-cancer cytostatic drugs.

A3RAgs were further found to be potent in inhibiting growth of tumorcells, other than lymphoma, e.g. melanoma or colon carcinoma (see forexample FIG. 6). A man versed in the art would clearly appreciate theadvantage of treating a subject with a non-specific anti-cancer drugcapable of inhibiting growth of the abnormally dividing cells whileconcomitantly being capable restoring the immune system of the subjectby inducing bone marrow cell proliferation.

FIGS. 7A–7B, for example, show the differential effect of A3RAg. In thisparticular case, the effect of IB-MECA, on tumor and normal cells wasevaluated. The more pronounced effect obtained using A3RAg, as comparedto adenosine, is also clearly presented by these results. Thetherapeutic effect of A3RAg was reversed when an A3 receptor antagonist,MRS-1220, was employed.

The in vivo studies confirmed the in vitro results which demonstrated achemoprotective effect of A3RAg on mice which were treatedsimultaneously with A3RAg and with a cytotoxic agent as compared to micetreated only with the cytotoxic drug (see for example FIG. 8). Further,a decrease in the number of foci in the A3RAg-treated mice was observedindicating the chemotherapeutic activity of A3RAg (see for example FIG.9). FIGS. 10A–10B as well as 19A and 19B, for example, show thattumor-bearing mice treated only with the cytotoxic drug exhibited adecline in the number of peripheral blood leukocytes and neutrophils,while administration of A3RAg after chemotherapy, resulted in therestoration of the total white blood cell count yielding an increase inthe percentage of neutrophils.

Thus, it may be concluded that A3RAg has a dual therapeutic function asit acts both as a chemotherapeutic agent as well as a chemoprotectiveagent. It is clear that use of A3RAg for this dual effect is also withinthe scope of the present invention.

In any case, the pharmaceutical compositions of the invention areadministered and dosed in accordance with good medical practice, takinginto account the clinical condition of the individual patient, the siteand method of administration, scheduling of administration, patient'sage, sex, body weight and other factors known to medical practitioners.

The composition of the invention may be administered in various ways. Itcan be administered orally, subcutaneously or parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally or byintranasal administration, as well as by intrathecal and infusiontechniques known to the man versed in the art.

As known, a treatment course in humans is usually longer than inanimals, e.g. mice, as exemplified herein. The treatment has a lengthproportional to the length of the disease process and active agenteffectiveness. The therapeutic regimen involved single doses or multipledoses over a period of several days or more. The treatment generally hasa length contingent with the course of the disease process, active agenteffectiveness and the patient species being treated.

When administering the compositions of the present inventionparenterally, it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion). The pharmaceuticalformulation suitable for injection includes sterile aqueous solutions ordispersions and sterile powders for reconstitution into sterileinjectable solutions or dispersions. The carrier employed can be asolvent or dispersing medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, lipid polyethyleneglycol and the like), suitable mixtures thereof and vegetable oils.

Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil,soybean oil, corn oil, sunflower oil, or peanut oil and ester, such asisopropyl myristate, may also at times be used as solvent systems forthe active ingredient.

Additionally, various additives which enhance the stability, sterilityand isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents and buffers can be added.Prevention of the action of microorganisms can be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid and the like.

For the purpose of oral administration, the active ingredient may beformulated in the form of tablets, suspensions, solutions, emulsions,capsules, powders, syrups and the like, are usable and may be obtainedby techniques well known to the pharmacists.

The present invention is defined by the claims, the contents of whichare to be read as included within the disclosure of the specification,and will now be described by way of example with reference to theaccompanying Figures. It is to be understood, that the terminology whichhas been used is intended to be in the nature of words of descriptionrather than limitation.

While the foregoing description describes in detail only a few specificembodiments of the invention, it will be understood by those skilled inthe art that the invention is not limited thereto and that othervariations in form and details may be possible without departing fromthe scope and spirit of the invention herein disclosed.

BRIEF DESCRIPTION OF THE FIGURES

In order to understand the invention and to see how it may be carriedout in practice, a preferred embodiment will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 is a bar graph showing results of an in vitro assay in which theeffect of adenosine (Ad), DPCPX (an A1RAn), CPA and CCPA (both A1RAg) orIB-MECA (an A3RAg) on G-CSF production is shown. Cultures treated withmodified RPMI served as the control. The results are presented in termsof percent of control (control=100%).

FIG. 2 is a bar graph showing results, obtained by [³]-thymidineincorporation assay, of an experiment in which stimulation ofproliferation of bone marrow cells by either adenosine, CPA or IB-MECA,with ((+) G-CSF Ab—light-colored columns) or without antibodies againstG-CSF ((−) G-CSF Ab—dark columns) was tested. The results show theneutralization effect of the anti-g-CSF antibodies. The results arerepresented in terms of percent increase over control (control=0%).

FIGS. 3A and 3B are two bar graphs showing results, obtained by a[³H]-thymidine incorporation assay, of an experiment, in whichproliferation of bone marrow cells was tested in the presence ofadenosine, an adenosine receptor agonists (FIG. 3A) or adenosine incombination with an adenosine receptor antagonists (FIG. 3B). Thereceptor agonists tested (FIG. 3A) are CPA (an A1RAg) and IB-MECA (anA3RAg); the receptor antagonists tested (FIG. 3B) were DPCPX (an A1RAn),DMPX (an A2RAn) and MRS (an A3RAn). The results are presented in termsof percent increase in thymidine incorporation over control(control=0%).

FIG. 4 is a bar graph showing results of an in vitro experiment in whichthe proliferation of bone marrow cells under three differentconcentrations of IB-MECA (0.01 μM, 0.1 μM and 1.0 μM) was tested. Theseresults are presented in terms of the [³H]-thymidineincorporation—percent above control (control 0%). The numbers below thebars are the IB-MCA concentrations (μM).

FIGS. 5A and 5B are bar graphs showing results of two experiments, bothcarried, out in vitro and being based on cell count assays, in which theeffect of growth of lymphoma cells (Nb2-11C) by adenosine and itsantagonist was tested. In the experiment shown in FIG. 5A, the effect onlymphoma cell growth by adenosine, CPA (an A1RAg), DMPA (an A2RAg) orIB-MECA (an A3RAg) was tested. In the experiment shown in FIG. 5B, theeffect on the lymphoma cell growth by adenosine, DPCPX (an A1RAn), DMPX(an A2RAn) or MRS-1220 (an A3RAn) was tested. RPMI-treated lymphomacells serve as control. The results are depicted as % inhibition ofgrowth over that of control (control=0%).

FIG. 6 is a bar graph showing results of an in vitro assay in whichgrowth of different tumor cell types (B16 melanoma, HTC-116 coloncarcinoma, Nb2-11C lymphoma) was inhibited in the presence of the A3RAgIB-MECA. RPMI-treated cells served as control. The results are presentedas percent inhibition over control (control=0%).

FIGS. 7A and 7B are bar graphs showing the results of an in vitro assayin which the effect of adenosine or the A3RAg, IB-MECA on growth oftumor cells (Nb2-11C Lymphoma, FIG. 7A) or bone marrow cells (FIG. 7Bwas tested). The results in FIGS. 7A and 7B are shown in terms ofpercent inhibition and percent stimulation, respectively, as compared tocontrol (control=0%).

FIG. 8 is a bar graph showing the results of an in vivo experiment wherethe count of peripheral white blood cells (WBC) after 5 and 9 days oftreatment with a chemotherapeutic drug (cyclophosphamide) was tested.The cyclophosphamide was either administered alone (gray columns) or incombination with IB-MECA administered orally (in a 1 ml solution) bydaily administration, beginning 24 hours after the chemotherapeuticdrug. PBS-treated mice served as control. The WBC count (WBC Counts) isgiven as percent over control (control=0%).

FIG. 9 is a bar graph showing results of an in vivo experiment in whichthe number of melanoma foci developed in mice following inoculation of2×10⁵ melanoma cells into the mice, treated with chemotherapycyclophosphamide (CHEMO), with IB-MECA, an A3RAg with a combination ofIB-MECA and CHEMO or with phosphate buffer saline (PBS) which served ascontrol.

FIGS. 10A and 10B are bar graphs showing the results of in vivoexperiment demonstrating the chemotherapeutic activity of IB-MECA. Thelevel of white blood cells (WBC, FIG. 10A) and neutrophils (FIG. 10B) asa function of time (hours after administration) of the chemotherapeuticdrug cyclophosphamide (CHEMO) with (CHEMO+IB-MECA) and without IB-MECAadministration is shown). Tumor bearing mice treated with PBS served ascontrol. The neutrophil count is shown as % over control (control=0%).

FIG. 11 shows weight of nude mice at 7, 10 and 14 days after onset oftreatment (administration of 5-FU, Cl-IB-MECA or a combination of 5-FUand Cl-IB-MECA), as % of control (non-treated mice=100%). The treatmentsconsisted of administration of 5-FU (dark columns), administration of5-FU in combination with Cl-IB-MECA (an A3RAg)—gray columns) andCl-IB-MECA alone (white columns).

FIGS. 12A and 12B show results of an experiment in which the effect ofCl-IB-MECA in reduction of doxorubicin-induced myelotoxicity wasexamined. The experiment was performed in ICR mice. FIG. 12A shows thewhite blood cell (WBC) count while FIG. 12B shows the count of bonemarrow nucleated cells. In FIG. 12A results are shown for the twodifferent treatments at four different time periods, with the controllevel being indicated by a dashed line, while in FIG. 12B, the resultsat two different time periods are shown with the control level beingrepresented by a bar at the left hand side.

FIG. 13 shows the effect of anti-G-CSF antibodies on the number of whiteblood cells (WBC) in control mice, mice treated with a chemotherapeuticdrug and mice treated with a chemotherapeutic drug and with Cl-IB-MECA,administered orally (6 μg/kg body weight, in 0.2 ml PBS). The number ofWBC following injection of anti-G-CSF antibodies is represented by thelight-colored columns. All results are presented as percent of control(control=100%).

FIG. 14 shows the size of tumor, over time, developed in nude micefollowing injection of HCT-116 human colon carcinoma cells, in a controlgroup and in a treated group (oral administration of Cl-IB-MECA).

FIG. 15 shows results of experiments similar to that of FIG. 14, wherethe size of the tumor developed in nude mice following injection ofHCT-116 human colon carcinoma cells was measured. Four groups weretested: a control group, a group receiving the chemotherapeutic drug5-FU, a group administered orally with Cl-IB-MECA and a group receivinga combined treatment of 5-FU and Cl-IB-MECA.

FIG. 16 is a bar graph showing the tumor size at day 30 in theexperiment depicted in FIG. 15.

FIG. 17 is a bar graph showing results of an experiment where theCl-IB-MECA-induced proliferation of bone marrow cells was measured underdifferent concentrations (0.05 μg/ml and 0.5 μg/ml) of anti-G-CSFantibodies (0—no antibodies). The proliferation was determined by[³H]-thymidine incorporation assay.

FIG. 18 shows results of an in vitro experiment where proliferation ofeither B-16 melanoma or bone marrow cells was measured. Theproliferation measured was the [³H]-thymidine incorporation assay. Thecells were exposed to either 0.01 μM and 0.1 μM Cl-IB-MECA with (whitecolumns) or without (dark columns) the A3RAg, MRS-1523. The results areshown in terms of percent of control (control=100%).

FIGS. 19A and 19B show results of an experiment similar to that shown inFIGS. 10A and 10B, respectively, performed with Cl-IB-MECA.

FIG. 20 shows results of an in vitro experiment in which theproliferation of bone marrow cells induced by IB-MECA or Cl-IB-MECA wasmeasured. These two A3RAg were added to the culture of the bone marrowcells at a concentration of either 1 nM or 10 nM, with (graycolumns—“(+) antagonists”) or without (dark columns—“(−) antagonists”).An A3RAn, MRS-1523, at a concentration of 10 nM. The proliferation wasdetermined by the [³H]-thymidine incorporation assay. The results aregiven as percent stimulation versus control (untreated bone marrowcells, control=0%).

EXPERIMENTAL RESULTS

Tumor Cells

Murine tumor cell lines (B-16 melanoma and Nb2 11c rat Lymphoma) wereused. B-16 melanoma cells were obtained from the American Type TissueCulture Collection (ATCC), Rockville, Md. Nb2-11C rat lymphoma cells[Pines M., and Gertler A. J. of Cellular Biochem., 37:119–129 (1988)]was kindly provided by Dr. A. Gertler, Hebrew university, Israel.

Colon carcinoma cells (HCT-116) were also employed and were obtained atthe ATCC.

The cells were routinely maintained in RPMI medium containing 10% fetalbovine serum (FBS, Biological Industries, Beit Haemek, Israel). Twice aweek the cells were transferred to a freshly prepared medium.

Normal Cells

Bone marrow cells derived from the femur of C57BL/6J mice were used. Thecells were prepared as previously described [17].

Drugs/Compounds

The drugs employed were: adenosine; adenosine A1 receptor agonists: CCPA[2-chloro-N⁶-cyclopentyl-adenosine], CPA (N-cyclopentyladenosine);A1RAn: DPCPX (1,3-dipropyl-8-cyclopentylxanthine); adenosine A2 receptoragonist: DMPA (N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) A2RAn: DMPX (3,7-dimethyl-1-propargyl-xantane); A3RAg:IB-MECA(1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuronamide)),CE-IB-MECA (2-chloro-N⁶-3-iodobenzyl)-adenosine-5′-N-methyl-uronamide;and adenosine A3 receptor antagonist: MRS-1523(5-propyl-2-ethyl-4-propyl-3-ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate)and MRS-1200 (9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4,]-triazolo[1,5-c]quinazoline).

Anti-murine G-CSF antibodies (rabbit antiserum purified by protein Achromatography, Cytolab LTD, Weizmann Institute of Science, Israel) wereused.

Cyclophosphamide was purchased from Taro Pharmaceutical Industries Ltd.Haifa Bay, Israel.

Mice

Female ICR, C57BL/6J or mice (BALB/C origin) mice aged 3 months,weighing an average of 25 g were used. The mice were purchased fromHarlan Laboratories, Jerusalem, ISRAEL. Standarized pelleted diet andtap water were supplied.

EXAMPLE 1 Effect of Adenosine and Adenosine Receptor Antagonists andAgonists on G-CSF Production and Bone Marrow Cell Proliferation

To test the assumption that adenosine exerts its biological effectthrough stimulation of G-CSF production, normal cells were cultured inthe presence adenosine or an adenosine agonist or antagonist.

For this purpose, bone marrow cells obtained from the femur of C57BL/6Jor ICR mice were first disaggregated by passing through a 25 G needle.Then, the cells (3×10⁵ cells/well, in 96 microtiter plates) wereincubated with RPMI medium containing 10% fetal bovine serum (FBS) inthe presence of adenosine (25 μM). Adenosine or agonists to the A1 andA3 adenosine receptors—CPA (an A1RAg, 0.01 μM), CCPA (an A1RAg, 0.01μM), or IB-MECA (an A3RAg, 0.01 μM), were added to the bone marrowcultures in the absence of adenosine; an A1 adenosine receptorantagonist, DPCPX (0.1 μM), was added to a bone marrow culture in thepresence of adenosine (25 μM).

Cultures containing cells suspended in RPMI medium and 5% FBS served asthe control for the above detailed experiment.

[³H]-Thymidine incorporation assay was used to evaluate theproliferation of the bone marrow cells. For this purpose, after 30 hoursof incubation, each well was pulsed with 1 μCi [³H]-Thymidine. After atotal of 48 hours of incubation, the cells were harvested and the[³H]-Thymidine uptake was determined in an LKB liquid scintillationcounter (LKB, Piscataway, N.J., USA). The results of this assay aredepicted in FIG. 1 which shows that A1RAg or A3RAg have an effect on theproduction of G-CSF, that is similar to that obtained with adenosine.

To confirm that adenosine and its agonists exert their effect viastimulation of G-CSF production, a further assay was conducted whereanti-G-CSF antibodies (62.5 ng/ml) were added to a culture of bonemarrow cells in the presence of adenosine (25 μM), CPA (0.01 μM) orIB-MECA (0.01 μM). Cell proliferation was evaluated as described above.The results of this experiment are depicted in FIG. 2 which shows thatantibodies to G-CSF inhibited the stimulatory effect of adenosine andits agonists on the proliferation of bone marrow cells. These resultssuggest that at least some of the activities associated with interactionwith adenosine receptors is mediated through the induction of G-CSF.

The cumulative effect on the proliferation of bone marrow cells, whenusing a combination of an A1RAgm1 A3RAg, (CPA and IB-MECA) wasevaluated. The assay was carried out similarly to that of the experimentthe results of which are shown in FIG. 1. Cells, after beingdisaggregated, were incubated in the presence of either adenosine (25μM), CPA (0.01 μM), IB-MECA (0.01 μM) or a combination of IB-MECA andCPA (each in a concentration of 0.01 μM) and further treated asdescribed above. The results are depicted in FIG. 3A which showsincreased combined effect of IB-MECA and CPA.

In order to compare the effect of adenosine receptor antagonist on theproliferation of bone marrow cells, following the same methodologydescribed above, cells were incubated with adenosine alone or incombination with either DMPX (an A2RAn), DPCPX (an A1RAn), MRS-1220 (anA3RAn) or with a combination of DPCPX and MRS-1220. The results areshown in FIG. 3B. As can be seen, blocking the A2 receptor by DMPX alsoresulted in an increased proliferation of bone marrow cells which evenexceeded that of adenosine alone. In comparison, proliferation withDPCPX or MRS-1220, reduced the increase by about 50% as compared toadenosine alone, while DPCPX in combination with MRS-1220 inhibitedproliferation altogether.

Cells pre-treated as described above, were incubated at differentconcentrations of IB-MECA (1 μM, 0.1 μM or 0.01 μM). The percent ofstimulation was determined by [³H]-Thymidine incorporation assay and theresults are depicted in FIG. 3 which show that IB-MECA stimulatesproliferation of bone marrow in a dose dependent manner.

EXAMPLE 2 Modulation of Tumor Cell Growth by Adenosine and its Agonists

Nb2-11C rat lymphoma cells (1.2×10⁴ cells/ml) were incubated for 48hours in 96 well microtiter plates with 1 ml RPMI medium containing 5%fetal bovine serum. Either 25 μM adenosine, 0.01 μM of an adenosinereceptor agonists (CPA, an A1RAg; DPMA, an A2RAg or IB-MECA, an A3RAg)or 0.1 μM of an adenosine receptor antagonists (DPCPX, an A1RAn; DMPX,an A2RAn; or MRS-1220, an A3RAn) in combination with adenosine (25 μM)was added.

Cultures containing cells suspended in RPMI medium with 5% FBS served ascontrols for the above detailed experiment. Extent of cell proliferationwas measured by a cell count assay.

The results are shown in FIGS. 5A and 5B, comparable to the inhibitionwith adenosine. As can be seen, the proliferation of Nb2-11C cells, wasmarkedly inhibited following incubation with IB-MECA, an A3RAg. Nogrowth inhibition was seen in the presence of CPA, an A1RAg, and a lowergrowth inhibition was seen in the presence of DPMA, an A2RAn. Thefailure of CPA to inhibit the proliferation of these two tumor cells,suggested that the adenosine A1 receptor is not involved in thisactivity. However, the inhibitory activity of both DMPA and IB-MECAsuggests the role of the A2 and the A3 adenosine receptors,respectively, in this inhibitory effect.

Further, it can be seen that DPCPX, an A1RAn, had essentially no effect,while in the presence of MRS-1220, an A3RAn, the effect of adenosine onthe proliferation of Nb2-11C cells was substantially abolished. A minor,however still significant effect was exerted by DMPX, an A2RAn. Thesefindings lead to the conclusion that tumor cell growth may beeffectively inhibited by an A3RAg or an A2RAn.

In the same manner as described above, inhibition of growth of B-16melanoma, HCT-116 colon carcinoma and Nb2-11C lymphoma, by the A3RAg,IB-MECA, was evaluated. The results are shown in FIG. 6 in terms ofpercent of inhibition or proliferation.

EXAMPLE 3 Adenosine A3 Receptor Agonists Exert a Differential Effect onTumor and Normal Cells

The effect of adenosine, A3RAns and A3RAgs, on the growth of tumor cellswas examined, following the experimental procedure described above.

Briefly, Nb2-11C lymphoma or bone marrow cells were incubated in thepresence of either adenosine, or IB-MECA. The dual effect of A3RAg isinhibiting the growth of tumor cells while stimulating the proliferationof bone marrow cells is depicted FIGS. 7A and 7B.

EXAMPLE 4 In vivo Studies

40 C57BL6/J mice were divided into 4 groups each of which were treated,by one of the following protocols:

-   -   1. Control group: daily intraperitoneal (i.p.) injection of 1 ml        saline per mouse from day of tumor inoculation until the mice        were sacrificed;    -   2. Chemotherapy group: one i.p. injection of cyclophosphamide 24        hours after inoculation of tumor cells and daily i.p. injection        of 1 ml saline per mouse from day of tumor inoculation until the        mice were sacrificed.    -   3. Adenosine A3 receptor agonist (A3RAg) group: daily oral        administration of IB-MECA from day of tumor inoculation until        the mice were sacrificed.    -   4. A3RAg+chemotherapy group: one i.p. injection of        cyclophosphamide 24 hours after inoculation of tumor cells and        daily oral administration of 3 μg/kg body weight of IB-MECA.

On day 5 and day 9 the mice were bled from the tail vain and bloodsamples were obtained for white blood cell (WBC) count. The results aredepicted in FIG. 8.

In addition, following 18 days the mice were sacrificed and melanomatumor foci were counted in the lung. The results are depicted in FIG. 9.

A further experiment was conducted in order to evaluate thechemoprotective effect of A3RAg. Mice were treated with cyclophosphamide(50 mg/kg body weight in 0.3 ml PBS). After 48 and 72 hours fromadministration of the cytotoxic drug, the mice were injected i.p. withadenosine (25 μg/kg body weight) or with IB-MECA (3 or 6 μg/kg bodyweight in 0.2 ml PBS). The number of white blood cells (WBC) andneutrophils was tested. The results are shown in FIGS. 10A (WBCs) and10B (neutrophils), respectively.

As can be seen, mice treated with cyclophosphamide only exhibited adecline in the number of peripheral blood leukocytes and neutrophils ascompared to the group treated only with IB-MECA. When adenosine orIB-MECA were administered, the total white blood cell count was restoredwith the latter having a very pronounced effect, yielding a completerecovery after 168 hours (7 days).

EXAMPLE 5 Adenosine A3 Receptor Agonist Prevents Weight Loss in MiceTreated with a Chemotherapeutic Drug

4 groups of nude mice (BALB/C origin), 10 in each group were treated asfollows:

-   -   Group 1: The mice were untreated [please confirm].    -   Group 2: The mice were injected intraperitoneally (i.p.) with        5-fluoro-uracyl (5-FU, 30 mg/kg body weight in PBS) for five        consecutive days.    -   Group 3: The mice were injected i.p. with 5-FU as in Group 2 but        starting on day 2, and every second day thereafter, the mice        were given an oral administration of Cl-IB-MECA (6 μg/kg body        weight, in 0.2 ml PBS.    -   Group 4: The mice received Cl-IB-MECA, as above.

The mice weight was measured at day 7, 10 and 14. The results are shownin FIG. 11.

As can be seen, 5-FU had a profound effect on the weight of the mice ascompared to control, while Cl-IB-MECA administered together with the5-FU, prevented some of this weight loss. The Cl-IB-MECA by itself didessentially not give rise to any weight loss.

This experiment demonstrates that the A3 adenosine receptor agonistshave general protecting effect on some of the toxic effects ofchemotherapy.

EXAMPLE 6 Cl-IB-MECA Protects the Mice Against Myelotoxic Effects of theChemotherapeutic Drug Doxorubicin

ICR mice were treated with doxorubicin (injection of 10 mg/kg i.p. in0.5 ml PBS). After 24, 48 and 72 hours from administration of thecytotoxic drug, the mice were orally administered with Cl-IB-MECA (6μg/kg body weight). At 72 hours, 96 hours, 120 hours and 144 hours, themice were sacrificed and blood samples were withdrawn. In addition, bonemarrow cells were aspirated from the femur of the mice and a cell countof nucleated cells from this aspirated preparation was made, followingstaining of the preparation with Coomassie Blue.

Three groups of mice were tested:

-   -   Group 1: (control) mice administered with PBS only.    -   Group 2: Mice treated with doxorubicin only.    -   Group 3: Administration of doxorubicin as above coupled with        administration of Cl-IB-MECA).

The results of the white blood cell count can be seen in FIG. 12A, andthat of the bone marrow nucleated cell count in FIG. 12B. These resultsclearly show that upon administration of Cl-IB-MECA, there is a markedincrease in the number of peripheral white blood cells as well as in thenumber of bone marrow nucleated cells. This is evident to the protectingeffect of the A3RAg against myelotoxic effects of doxorubicin.

EXAMPLE 7 Antibodies Against G-CSF Neutralize the MyeloprotectionalEffect of Cl-IB-MECA

ICR mice, were divided into six groups as follows:

-   -   Group 1: Control—administration of the vehicle only.    -   Group 2: Control with anti-G-CSF antibodies (5 μg/mouse).    -   Group 3: Chemotherapy—administration of cyclophosphamide CYP—50        mg/kg body weight).    -   Group 4: Chemotherapy (50 mg/kg body weight CYP)+anti-G-CSF        antibodies (5 μg/mouse).    -   Group 5: Chemotherapy (50 mg/kg body weight CYP)+Cl-IB-MECA (6        μg/kg body weight)+anti-G-CSF antibodies (5 μg/mouse).    -   Group 6: Chemotherapy (50 mg/kg body weight CYP)+Cl-IB-MECA (6        μg/kg body weight)+anti-G-CSF antibodies (5 μg/mouse).

Each group consisted of 10 mice and the experiment was repeated twice.

The CYP was injected intraperitoneally in 0.2 ml of PBS which served asthe carrier.

Cl-IB-MECA was given orally (in 0.2 ml PBS) at 48 hours and 72 hoursfollowing the administration of the cyclophosphamide.

Anti-G-CSF antibodies were intravenously injected (in 0.2 ml PBS) 72hours following the administration of the chemotherapeutic drugs.

Blood samples were withdrawn 124 hours following chemotherapy. Whiteblood cells (WBC) counts were made in a Coulter counter and differentialcell counts were carried on smear preparations stained withMay-Grundvald-Giemsa solution.

The results of the WBC count is shown in FIG. 13. As can be seen, micetreated with cyclophosphamide only showed a decline in the number ofperipheral blood WBC. In the group that was treated with Cl-IB-MECA, theWBC counts and the percentage of neutrophils were significantly higherin comparison to the chemotherapeutic treated group (results regardingtransfer of neutrophils not shown). When anti-G-CSF antibodies wereadministered to the control or the chemotherapy groups, an expecteddecline in the number of WBC was observed. Administration of anti-G-CSFantibodies to the mice treated with the combination of thechemotherapeutic drug and Cl-IB-MECA, cancelled the protective effect ofCl-IB-MECA, as can clearly be seen in FIG. 13. These results lead to theconclusion that the protective effect of Cl-IB-MECA on the myeloidsystem is mediated through the ability of a Cl-IB-MECA to promote theproduction and secretion of G-CSF.

EXAMPLE 8 Cl-IB-MECA Inhibits the Development of HCT-116 Human CoonCarcinoma in Nude Mice

Tumors were established by subcutaneous injection of 1×10⁶ HCT-116 humancolon cancer cells to nude mice (BALB/C origin) (Harlan, Jerusalem,Israel). Mice were treated orally with 6 μg/kg body weight Cl-IB-MECA(in 0.2 ml of PBS) every other day. Mice that were treated with thevehicle only (PBS). Each group consisted of 10 mice. Tumor growth ratewas determined by measuring two orthogonal diameters of each tumor twicea week, and the tumor size was estimated according to the followingformula: π/6[D₁D₂]. The results are depicted in FIG. 14. As can be seen,in the treated group there is a marked inhibition of tumor growth.

In a separate set of experiments a combined therapy of Cl-IB-MECA and5-fluorouracyl (5-FU) was tested. 1×10⁶ HCT-116 cells were injectedsubcutaneously to nude mice. One day later, 5-FU (30 mg/kg body weight,in 0.2 ml PBS) was intraperitoneally injected and subsequently in 4additional consecutive days. Every other day, the mice were administeredorally with 5 μg/kg body weight of Cl-IB-MECA (in 0.2 ml of PBS). Micethat were treated either with the vehicle only (PBS) or with 5-FU servedas control. Each group consisted of 10 mice. Tumor growth rate wasdetermined by measuring two orthogonal diameters of each tumor twice aweek, and the tumor size was estimated according to the followingformula: π/6[D₁D₂].

The results are depicted in FIGS. 15 and 16. A marked inhibition oftumor growth was observed in the groups treated with 5-FU, Cl-IB-MECAand the combined therapy of Cl-IB-MECA and 5-FU. After 20 days a clearsynergistic effect between Cl-IB-MECA and 5-FU in noting the tumor masswas seen, as depicted particularly in FIG. 16 (the results representedin FIG. 16 are those at day 30).

EXAMPLE 9 Cl-IB-MECA Stimulates Bone Marrow Cell Proliferation Throughthe Induction of G-CSF Production

Bone marrow cells (3×10⁶ cell/ml) were incubated in wells of 96microtiter plates. Cl-IB-MECA at a final concentration of 10 nM wasadded with or without anti-G-CSF antibodies, at a final concentration of0.05 and 0.5 μg/ml. Cell proliferation was measured by [³H]-thymidineincorporation assay. The results are shown in FIG. 17.

As can be seen, the anti-G-CSF antibodies inhibit the proliferation ofthe bone marrow cells in a dose-dependent manner. This experiment alsoshows that the action of Cl-IB-MECA is mediated through the G-CSFpathway (involving G-CSF secretion from the cells).

EXAMPLE 10 Cl-IB-MECA Inhibits Tumor Cell Growth and Stimulates BoneMarrow Proliferation and Differentiation

B-16 melanoma cells (5×10⁵ cells/ml) and bone marrow cells (3×10⁶cells/ml) were incubated in wells of 96 microtiter plate. The cultureconsisted of RPMI medium supplemented with 10% FTS: Cl-IB-MECA, at theconcentration of 0.01 μM or 0.1 μM was added, with or without anantagonist of the adenosine A3 receptor, MRS-1523. Cell proliferationwas measured by the [³H]-thymidine incorporation assay mentioned before.The results are shown in FIG. 18. As can be seen, in the presence ofMRS-1523, the proliferation of both the B-16 melanoma cells and the bonemarrow cells was unchanged versus control. Against this, the Cl-IB-MECAexerted an inhibitory effect on proliferation of the B-16 melanomacells, and a proliferation stimulation effect on the bone marrow cells.

These results demonstrate the dual effect of the A3 adenosine receptoragonists.

EXAMPLE 11 Cl-IB-MECA Acts as a Chemoprotective Agent

An example similar to that of Example 4, was performed with Cl-IB-MECAand the results are shown in FIGS. 19A and 19B demonstrating thechemoprotective activity of Cl-IB-MECA.

EXAMPLE 12 Effect of IB-MECA and Cl-IB-MECA on the Proliferation of Bonemarrow cells

Murine bone marrow cells were cultured as described above. IB-MCA orCl-IB-MECA were added to the cultures at a concentration of 1 or 10 nM,in the presence or absence of the A3RAn, MRS-1523. The antagonist wasadded at a concentration of 10 nM. The results are shown in FIG. 20.

As can be seen in FIG. 20, the effect of both IB-MECA and Cl-IB-MECA isdose dependent. Furthermore, as can also be seen, this effect isinhibited to a large extent by the A3RAn.

1. A method for selectively inhibiting abnormal cell proliferation in asubject in need thereof, comprising administering to the subject anamount of an A3-selective adenosine A3 receptor agonist (A3RAg), in amanner such that it exerts is prime effect through the adenosine A3receptor, the amount being less than 100 μg/Kg body weight.
 2. A methodaccording to claim 1, for inhibiting growth or proliferation of tumorcells.
 3. A method according to claim 1, wherein the drug isadministered orally.
 4. A method according to claim 1, wherein the drugis administered in combination with a chemotherapeutic drug.
 5. A methodaccording to claim 1, wherein said active ingredient is an A3-selectiveA3RAg that is a nucleoside derivative of the following general formula(I):

wherein R₁ is C₁–C₁₀ alkyl, C₁–C₁₀ hydroxyalkyl, C₁–C₁₀ carboxyalkyl orC₁–C₁₀ cyanoalkyl or a group of the following general formula (II):

in which: Y is an oxygen or sulfur atom or CH₂; X₁ is H, C₁–C₁₀ alkyl,R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein R^(a) and R^(b) may be the sameor different and are selected from the group consisting of hydrogen,C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀BOC-aminoalkyl, and C₃–C₁₀ cycloalkyl or are joined together to form aheterocyclic ring containing two to five carbon atoms, and R^(c) isselected from the group consisting of C₁–C₁₀ alkyl, amino, C₁–C₁₀haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀ BOC-aminoalkyl, and C₃–C₁₀cycloalkyl; X₂ is H, hydroxyl, C₁–C₁₀ alkylamino, C₁–C₁₀ alkylamido orC₁–C₁₀ hydroxyalkyl; X₃ and X₄ each independently are hydrogen,hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo,nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, —OCOPh,—OC(═S)OPh or both X₃ and X₄ are oxygen connected to >C═S to form a5-membered ring, or X₂ and X₃ form the ring of formula (III):

where R′ and R″ are independently C₁–C₁₀ alkyl; R₂ is selected from thegroup consisting of hydrogen, halo, C₁–C₁₀ alkylether, amino, hydrazido,C₁–C₁₀ alkylamino, C₁–C₁₀ alkoxy, C₁–C₁₀ thioalkoxy, pyridylthio, C₂–C₁₀alkenyl; C₂–C₁₀ alkynyl, thio, and C₁–C₁₀ alkylthio; and R₃ is a —NR₄R₅group with R₄ being hydrogen, alkyl, substituted alkyl or aryl-NH—C(Z)—,with Z being O, S or NR^(a), and, when R₄ is hydrogen, R₅ being selectedfrom the group consisting of R- and S-1-phenylethyl, benzyl, phenylethylor anilide groups, each said group being unsubstituted or substituted inone or more positions with a substituent selected from the groupconsisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl, nitro,hydroxyl, acetamido, C₁–C₁₀ alkoxy, and sulfonic acid or a salt thereof;or R₅ being benzodioxanemethyl, fururyl, L-propylalanylaminobenzyl,β-alanylaminobenzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl,phenoxy or C₁–C₁₀ cycloalkyl; or R₅ being a group of the followingformula:

or, when R₄ is alkyl, substituted alkyl, or aryl-NH—C(Z)—, then R₅ beingselected from the group consisting of substituted or unsubstitutedheteroaryl-NR^(a)—C(Z), heteroaryl-C(Z)—, alkaryl-NR^(a)—C(Z)—,alkaryl-C(Z)—, aryl-NR—C(Z)— and aryl-C(Z); or a suitable salt of thecompound defined above.
 6. A method according to claim 5, wherein saidactive ingredient is an A3-selective A3RAg that is a nucleosidederivative of the general formula (IV):

in which X₁, R₂ and R₅ are as defined in claim
 5. 7. A method accordingto claim 6, wherein said active ingredient is anN⁶-benzyladenosine-5′-uronamide.
 8. A method according to claim 7,wherein said active ingredient is selected from the group consisting ofN⁶-2-(4-aminophenyl) ethyladenosine (APNEA),N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide) (AB-MECA) and1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuronamide (IB-MECA) and2-chloro-N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (Cl-IB-MECA).9. A method for treating cancer in a subject in need thereof, whichsubject is undergoing chemotherapeutic drug treatment, comprisingadministering to the subject an amount of an A3-selective adenosine A3receptor agonist (A3RAg), in a manner such that in exerts its primeeffect through the adenosine A3 receptor, the amount being effective toboth selectively inhibit proliferation of cancer cells and to countertoxic side effects of chemotherapeutic drug treatment of the samesubject, wherein said amount is less than 100 μg/Kg body weight.
 10. Amethod according to claim 9, wherein the A3RAg synergizes with saidchemotherapeutic drug to yield a stronger anti-tumor effect.
 11. Amethod according to claim 9, wherein the drug is administered orally.12. A method according to 9, wherein said active ingredient is anA3-selective A3RAg that is a nucleoside derivative of the followinggeneral formula (I):

wherein R₁ is C₁–C₁₀ alkyl, C₁–C₁₀ hydroxyalkyl, C₁–C₁₀ carboxyalkyl orC₁–C₁₀ cyanoalkyl or a group of the following general formula (II):

in which: Y is an oxygen or sulfur atom or CH₂; X₁ is H, C₁–C₁₀ alkyl,R^(a) R^(b) NC(═O)— or HOR^(c)—, wherein R^(a) and R^(b) may be the sameor different and are selected from the group consisting of hydrogen,C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀BOC-aminoalkyl, and C₃–C₁₀ cycloalkyl or are joined together to form aheterocyclic ring containing two to five carbon atoms, and R^(c) isselected from the group consisting of C₁–C₁₀ alkyl, amino, C₁–C₁₀haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀ BOC-aminoalkyl, and C₃–C₁₀cycloalkyl; X₂ is H, hydroxyl, C₁–C₁₀ alkylamino, C₁–C₁₀ alkylamido orC₁–C₁₀ hydroxyalkyl; X₃ and X₄ each independently are hydrogen,hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo,nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, —OCOPh,—OC(═S)OPh or both X₃ and X₄ are oxygen connected to >C═S to form a5-membered ring, or X₂ and X₃ form the ring of formula (III):

where R′ and R″ are independently C₁–C₁₀ alkyl; R₂ is selected from thegroup consisting of hydrogen, halo, C₁–C₁₀ alkylether, amino, hydrazido,C₁–C₁₀ alkylamino, C₁–C₁₀ alkoxy, C₁–C₁₀ thioalkoxy, pyridylthio, C₂–C₁₀alkenyl; C₂–C₁₀ alkynyl, thio, and C₁–C₁₀ alkylthio; and R₃ is a —NR₄R₅group with R₄ being hydrogen, alkyl, substituted alkyl or aryl-NH—C(Z)—,with Z being O, S or NR^(a), and, when R₄ is hydrogen, R₅ being selectedfrom the group consisting of R- and S-1-phenylethyl, benzyl, phenylethylor anilide groups, each said group being unsubstituted or substituted inone or more positions with a substituent selected from the groupconsisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl, nitro,hydroxyl, acetamido, C₁–C₁₀ alkoxy, and sulfonic acid or a salt thereof;or R₅ being benzodioxanemethyl, fururyl, L-propylalanylaminobenzyl,β-alanylaminobenzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl,phenoxy or C₁–C₁₀ cycloalkyl; or R₅ being a group of the followingformula:

or, when R₄ is alkyl, substituted alkyl, or aryl-NH—C(Z)—, then R₅ beingselected from the group consisting of substituted or unsubstitutedheteroaryl-NR^(a)—C(Z), heteroaryl-C(Z)—, alkaryl-NR^(a)—C(Z)—,alkaryl-C(Z)—, aryl-NR—C(Z)— and aryl-C(Z); or a suitable salt of thecompound defined above.
 13. A method according to claim 12, wherein saidactive ingredient is an A3-selective A3RAg that is a nucleosidederivative of the general formula (IV):

in which X₁, R₂ and R₅ are as defined in claim
 12. 14. A methodaccording to claim 13, wherein said active ingredient is anN⁶-benzyladenosine-5′-uronamide.
 15. A method according to claim 14,wherein said active ingredient is selected from the group consisting ofN⁶-2-(4-aminophenyl)ethyladenosine (APNEA),N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide) (AB-MECA) and1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}N-methyl-β-D-ribofuranuronamide(IB-MECA) and 2- chloro-N⁶(3-iodobenzyl)adenosine-5′-N-methyluronamide(Cl-IB-(MECA).
 16. A method for selectively inhibiting abnormal cellproliferation in a subject, comprising administering to the subject anamount of an adenosine A3 receptor agonist (A3RAg) in a manner such thatit exerts its prime effect through the A3 adenosine receptor withoutessentially activating adenosine receptors other than the A3 adenosinereceptor, the amount being less than 100 μg/Kg body weight.
 17. A methodaccording to claim 16, wherein said abnormal cell proliferation is thegrowth or proliferation of tumor cells.
 18. A method according to claim16, wherein the drug is administered orally.
 19. A method according toclaim 16, wherein the drug is administered in combination with achemotherapeutic drug.
 20. A method according to claim 16, wherein theactive ingredient is an A3RAg that exerts its prime effect through theA3 adenosine receptor without essentially activating adenosine receptorsother then the A3 adenosine receptor, which is a nucleoside derivativeof general formula (I):

wherein R₁ is C₁–C₁₀ alkyl, C₁–C₁₀ hydroxyalkyl, C₁–C₁₀ carboxyalkyl orC₁–C₁₀ cyanoalkyl or a group of the following general formula (II):

in which: Y is an oxygen or sulfur atom or CH₂; X₁ is H, C₁–C₁₀ alkyl,R^(a)R^(b)NC(═O)— or HOR^(c)—, wherein R^(a) and R^(b) may be the sameor different and are selected from the group consisting of hydrogen,C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀BOC-aminoalkyl, and C₃–C₁₀ cycloalkyl or are joined together to form aheterocyclic ring containing two to five carbon atoms, and R^(c) isselected from the group consisting of C₁–C₁₀ alkyl, amino, C₁–C₁₀haloalkyl, C₁–C₁₀ aminoalkyl, C₁–C₁₀ BOC-aminoalkyl, and C₃–C₁₀cycloalkyl; X₂ is H, hydroxyl, C₁–C₁₀ alkylamino, C₁–C₁₀ alkylamido orC₁–C₁₀ hydroxyalkyl; X₃ and X₄ each independently are hydrogen,hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo,nitro, trifluoro, aryl, alkaryl, thio, thioester, thioether, —OCOPh,—OC(═S)OPh or both X₃ and X₄ are oxygen connected to >C═S to form a5-membered ring, or X₂ and X₃ form the ring of formula (III):

where R′ and R″ are independently C₁–C₁₀ alkyl; R₂ is selected from thegroup consisting of hydrogen, halo, C₁–C₁₀ alkylether, amino, hydrazido,C₁–C₁₀ alkylamino, C₁–C₁₀ alkoxy, C₁–C₁₀ thioalkoxy, pyridylthio, C₂–C₁₀alkenyl, C₂–C₁₀ alkynyl, thio, and C₁–C₁₀ alkylthio; and R₃ is a —NR₄R₅group with R₄ being hydrogen, alkyl, substituted alkyl or aryl-NH—C(Z)—,with Z being O, S or NR^(a), and, when R₄ is hydrogen, R₅ being selectedfrom the group consisting of R- and S-1-phenylethyl, benzyl, phenylethylor anilide groups, each said group being unsubstituted or substituted inone or more positions with a substituent selected from the groupconsisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl, nitro,hydroxyl, acetamido, C₁–C₁₀ alkoxy, and sulfonic acid or a salt thereof;or R₅ being benzodioxanemethyl, fururyl, L-propylalanyl-aminobenzyl,β-alanylamino-benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl,phenoxy or C₁–C₁₀ cycloalkyl; or R₅ being a group of the followingformula:

or, when R₄ is alkyl, substituted alkyl, or aryl-NH—C(Z)—, then R₅ isselected from the group consisting of substituted or unsubstitutedheteroaryl-NR^(a)—C(Z)—, heteroaryl-C(Z)—, alkaryl-NR^(a)—C(Z)—,alkaryl-C(Z)—, aryl-NR—C(Z)— and aryl-C(Z)—; or a suitable salt of saidnucleotide derivative.
 21. A method according to claim 20, wherein saidactive ingredient is an A3RAg that exerts its prime effect through theA3 adenosine receptor without essentially activating adenosine receptorsother then the A3 adenosine receptor, which is a nucleoside derivativeof the general formula (IV):

in which X₁, R₂ and R₄ are as defined in claim
 20. 22. A methodaccording to claim 21, wherein said active ingredient is anN⁶⁻benzyladenosine-5′-uronamide.
 23. A method according to claim 22,wherein said active ingredient is selected from the group consisting ofN⁶-2-(4-aminophenyl)ethyladenosine (APNEA), N⁶-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide) (AB-MECA) and1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purine-9-yl}-N-methyl-β-D-ribofuranuron-amide(IB-MECA) and 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methly-uronamide(Cl-IB-MECA).
 24. A method according to claim 16, wherein the amount isless than 50 μg/Kg body weight.
 25. A method according to claim 20,wherein said active ingredient is selected from the group consisting of:N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-hydroxyethyladenine;R—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;S—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;N⁶-(3-iodobenzyladenin-9-yl)acetic acid;N⁶-(3-iodobenzyl)-9-(3-cyanopropyl)adenine;2-chloro-N⁶-(3-iodobenzyl)-9-methyladenine;2-amino-N⁶-(3-iodobenzyl)-9-methyladenine;2-hydrazido-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methylamino-9-methyladenine;2-dimethylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-propylaminoadenine;2-hexylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methoxy-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-methylthioadenine;N⁶-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine;(1S,2R,3S,4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2,3-triol;(1S,2R,3S,4R)-4-(6-amino-2-chloro-9H-purin-9-yl)cyclopentane-1,2,3-triol;(±)-9-[2α,3α-dihydroxy-4β-(N-methylcarbamoyl)cyclopent-1β-yl)]-N⁶-(3-iodobenzyl)-adenine;2-chloro-9-(2′-amino-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(2′,3′-dideoxy-2′-fluoro-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;9-(2-acetyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-2-methanesulfonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-β-D-5-ribofuranosyl)—N⁶—(3-iodobenzyl) adenine;2-chloro-9-(2′,3′-O-thiocarbonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;9-(2-phenoxythiocarbonyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-β-D-ribofuranosiduronamide;2-chloro-9-(2,3-dideoxy-β-D-5-methyl-ribofuronamido) —N⁶-benzyladenine;2-chloro-9-(2′-azido-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-benzyladenine;2-chloro-9-(β-D-erythrofuranoside)-N⁶-(3-iodobenzyl)adenine;N⁶-(benzodioxanemethyl)adenosine;1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-β-D-ribofuranosiduronamide;N⁶-[3-(L-prolylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(β-alanylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(N—T-Boc-β-alanylamino)benzyl]adenosine-5′-N-methyluronamide6-(N′-phenylhydrazinyl)purine-9-β-ribofuranoside-5′-N-methyluronamide;6-(O-phenylhydroxylamino)purine-9-β-ribofuranoside-5′-N-methyluronamide;9-(β-D-2′,3′-dideoxyerythrofuranosyl)-N⁶-[(3-β-alanylamino)benzyl]adenosine;9-(β-D-erythrofuranoside)-2-methylamino-N⁶-(3-iodobenzyl)adenine;2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine;2-chloro-(2′-deoxy-6′-thio-L-arabinosyl)adenine;2-chloro-(6′-thio-L-arabinosyl)adenine;N⁶-(4-biphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-methoxyphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-chlorophenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(phenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(benzylcarbamoylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-sulfonamido-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-acetyl-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((R)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((S)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-n-propoxy-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-bis-(4-nitrophenylcarbamoyl)-adenosine-5′-N-ethyluronamide; andN⁶-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide.26. A method according to claim 21, wherein said active ingredient is anA3 selective A3RAg that is selected from the group consisting of thoseof formula (IV) in which: X₁ is R^(a)R^(b) NC(═O), wherein R^(a) andR^(b) may be the same or different and are selected from the groupconsisting of hydrogen, C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀aminoalkyl, and C₃–C₁₀ cycloalkyl, R₂ is selected from the groupconsisting of hydrogen, halo, C₁–C₁₀ alkyoxy, amino, C₂–C₁₀ alkenyl, andC₂–C₁₀ alkynyl, and R₅ is selected from the group consisting of R- andS-1-phenylethyl, an unsubstituted benzyl group, and a benzyl groupsubstituted in one or more positions with a substituent selected fromthe group consisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl,nitro, hydroxy, acetamido, C₁–C₁₀ alkoxy, and sulfo.
 27. A methodaccording to claim 26, wherein said active ingredient is an A3 selectiveA3RAg that is selected from the group consisting of those of formula(IV) in which: R^(a) and R^(b) are the same or different and areselected from the group consisting of hydrogen and C₁–C₁₀ alkyl, and R₂is hydrogen or halo; R^(a) is hydrogen, R₂ is hydrogen and R₅ isunsubstituted benzyl; R^(b) is C₁–C₁₀ alkyl or C₃–C₁₀ cycloalkyl and R₅in R- or S-1-phenylethyl or a benzyl substituted in one or morepositions with a substituent selected from the group consisting of halo,amino, acetamido, C₁–C₁₀ haloalkyl and sulfo, wherein the sulfoderivative is a salt; R₂ is a C₂–C₁₀ alkyne of the formula R^(d)—C═C—where R^(d) is a C₁–C₈ alkyl; or R₂ is a halo, C₁–C₁₀ alkylamino, orC₁–C₁₀ alkylthio, R^(a) is hydrogen, R^(b) is C₁–C₁₀ alkyl and R₅ is asubstituted benzyl.
 28. A method according to claim 20, wherein theactive ingredient is an A3 selective A3RAg that is in the form of atriethylammonium salt.
 29. A method according to claim 5, wherein saidactive ingredient is selected from the group consisting of:N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-hydroxyethyladenine;R—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;S—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;N⁶-(3-iodobenzyladenin-9-yl)acetic acid;N⁶-(3-iodobenzyl)-9-(3-cyanopropyl)adenine;2-chloro-N⁶-(3-iodobenzyl)-9-methyladenine;2-amino-N⁶-(3-iodobenzyl)-9-methyladenine;2-hydrazido-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methylamino-9-methyladenine;2-dimethylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-propylaminoadenine;2-hexylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methoxy-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-methylthioadenine;N⁶-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine;(1S,2R,3S,4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2,3-triol;(1S,2R,3S,4R)-4-(6-amino-2-chloro-9H-purin-9-yl)cyclopentane-1,2,3-triol;(±)-9-[2α,3α-dihydroxy-4β-(N-methylcarbamoyl)cyclopent-1β-yl)]-N⁶-(3-iodobenzyl)-adenine;2-chloro-9-(2′-amino-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(2′,3′-dideoxy-2′-fluoro-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;9-(2-acetyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-2-methanesulfonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-β-D-5-ribofuranosyl)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(2′,3′-O-thiocarbonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;9-(2-phenoxythiocarbonyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-β-D-ribofuranosiduronamide;2-chloro-9-(2,3-dideoxy-β-D-5-methyl-ribofuronamido)-N⁶-benzyladenine;2-chloro-9-(2′-azido-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-benzyladenine;2-chloro-9-(β-D-erythrofuranoside)-N⁶-(3-iodobenzyl)adenine;N⁶-(benzodioxanemethyl)adenosine;1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-β-D-ribofuranosiduronamide;N⁶-[3-(L-prolylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(β-alanylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(N—T-Boc-β-alanylamino)benzyl]adenosine-5′-N-methyluronamide6-(N′-phenylhydrazinyl)purine-9-β-ribofuranoside-5′-N-methyluronamide;6-(O-phenylhydroxylamino)purine-9-β-ribofuranoside-5′-N-methyluronamide;9-(β-D-2′,3′-dideoxyerythrofuranosyl)-N⁶-[(3-β-alanylamino)benzyl]adenosine;9-(β-D-erythrofuranoside)-2-methylamino-N⁶-(3-iodobenzyl)adenine;2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine;2-chloro-(2′-deoxy-6′-thio-L-arabinosyl)adenine;2-chloro-(6′-thio-L-arabinosyl)adenine;N⁶-(4-biphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-methoxyphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-chlorophenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(phenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(benzylcarbamoylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-sulfonamido-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-acetyl-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((R)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((S)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-n-propoxy-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-bis-(4-nitrophenylcarbamoyl)-adenosine-5′-N-ethyluronamide; andN⁶-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide.30. A method according to claim 12, wherein said active ingredient isselected from the group consisting of:N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-hydroxyethyladenine;R—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;S—N⁶-(3-iodobenzyl)-9-(2,3-dihydroxypropyl)adenine;N⁶-(3-iodobenzyladenin-9-yl)acetic acid;N⁶-(3-iodobenzyl)-9-(3-cyanopropyl)adenine;2-chloro-N⁶-(3-iodobenzyl)-9-methyladenine;2-amino-N⁶-(3-iodobenzyl)-9-methyladenine;2-hydrazido-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methylamino-9-methyladenine;2-dimethylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-propylaminoadenine;2-hexylamino-N⁶-(3-iodobenzyl)-9-methyladenine;N⁶-(3-iodobenzyl)-2-methoxy-9-methyladenine;N⁶-(3-iodobenzyl)-9-methyl-2-methylthioadenine;N⁶-(3-iodobenzyl)-9-methyl-2-(4-pyridylthio)adenine;(1S,2R,3S,4R)-4-(6-amino-2-phenylethylamino-9H-purin-9-yl)cyclopentane-1,2,3-triol;(1S,2R,3S,4R)-4-(6-amino-2-chloro-9H-purin-9-yl)cyclopentane-1,2,3-triol;(±)-9-[2α,3α-dihydroxy-4β-(N-methylcarbamoyl)cyclopent-1β-yl)]-N⁶-(3-iodobenzyl)-adenine;2-chloro-9-(2′-amino-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(2′,3′-dideoxy-2′-fluoro-β-D-5′-methyl-arabino-furonamido)-N⁶-(3-iodobenzyl)adenine;9-(2-acetyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-2-methanesulfonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3-deoxy-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(3,5-1,1,3,3-tetraisopropyldisiloxyl-β-D-5-ribofuranosyl)-N⁶-(3-iodobenzyl)adenine;2-chloro-9-(2′,3′-O-thiocarbonyl-β-D-5-methyl-ribofuronamido)-N⁶-(3-iodobenzyl)adenine;9-(2-phenoxythiocarbonyl-3-deoxy-β-D-5-methyl-ribofuronamido)-2-chloro-N⁶-(3-iodobenzyl)adenine;1-(6-benzylamino-9H-purin-9-yl)-1-deoxy-N,4-dimethyl-β-D-ribofuranosiduronamide;2-chloro-9-(2,3-dideoxy-β-D-5-methyl-ribofuronamido)-N⁶-benzyladenine;2-chloro-9-(2′-azido-2′,3′-dideoxy-β-D-5′-methyl-arabino-furonamido)-N⁶-benzyladenine;2-chloro-9-(β-D-erythrofuranoside)-N⁶-(3-iodobenzyl)adenine;N⁶-(benzodioxanemethyl)adenosine;1-(6-furfurylamino-9H-purin-9-yl)-1-deoxy-N-methyl-β-D-ribofuranosiduronamide;N⁶-[3-(L-prolylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(β-alanylamino)benzyl]adenosine-5′-N-methyluronamide;N⁶-[3-(N—T-Boc-β-alanylamino)benzyl]adenosine-5′-N-methyluronamide6-(N′-phenylhydrazinyl)purine-9-β-ribofuranoside-5′-N-methyluronamide;6-(O-phenylhydroxylamino)purine-9-β-ribofuranoside-5′-N-methyluronamide;9-(β-D-2′,3′-dideoxyerythrofuranosyl)-N⁶-[(3-β-alanylamino)benzyl]adenosine;9-(β-D-erythrofuranoside)-2-methylamino-N⁶-(3-iodobenzyl)adenine;2-chloro-N-(3-iodobenzyl)-9-(2-tetrahydrofuryl)-9H-purin-6-amine;2-chloro-(2′-deoxy-6′-thio-L-arabinosyl)adenine;2-chloro-(6′-thio-L-arabinosyl)adenine;N⁶-(4-biphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(2,4-dichlorobenzyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-methoxyphenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-chlorophenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(phenyl-carbonylamino)-adenosine-5′-N-ethyluronamide;N⁶-(benzylcarbamoylamino)-adenosine-5′-N-ethyluronamide;N⁶-(4-sulfonamido-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-acetyl-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((R)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-((S)-α-phenylethylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(5-methyl-isoxazol-3-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(1,3,4-thiadiazol-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-(4-n-propoxy-phenylcarbamoyl)-adenosine-5′-N-ethyluronamide;N⁶-bis-(4-nitrophenylcarbamoyl)-adenosine-5′-N-ethyluronamide; andN⁶-bis-(5-chloro-pyridin-2-yl-carbamoyl)-adenosine-5′-N-ethyluronamide.31. A method according to claim 5, wherein the active ingredient is anA3 selective A3RAg that is in the form of a triethylammonium salt.
 32. Amethod according to claim 12, wherein the active ingredient is an A3selective A3RAg that is in the form of a triethylammonium salt.
 33. Amethod according to claim 6, wherein said active ingredient is an A3selective A3RAg that is selected from the group consisting of those offormula (IV) in which: X₁ is R^(a)R^(b)NC(=O), wherein R^(a) and R^(b)may be the same or different and are selected from the group consistingof hydrogen, C₁–C₁₀ alkyl, amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl,and C₃–C₁₀ cycloalkyl, R₂ is selected from the group consisting ofhydrogen, halo, C₁–C₁₀ alkyoxy, amino, C₂–C₁₀ alkenyl, and C₂–C₁₀alkynyl, and R₄ is selected from the group consisting of R- andS-1-phenylethyl, an unsubstituted benzyl group, and a benzyl groupsubstituted in one or more positions with a substituent selected fromthe group consisting of C₁–C₁₀ alkyl, amino, halo, C₁–C₁₀ haloalkyl,nitro, hydroxy, acetamido, C₁–C₁₀ alkoxy, and sulfo.
 34. A methodaccording to claim 33, wherein said active ingredient is an A3 selectiveA3RAg that is selected from the group consisting of those of formula(IV) in which: R^(a) and R^(b) are the same or different and areselected from the group consisting of hydrogen and C₁–C₁₀ alkyl, and R₂is hydrogen or halo; R^(a) is hydrogen, R₂ is hydrogen and R₅ isunsubstituted benzyl; R^(b) is C₁–C₁₀ alkyl or C₃–C₁₀ cycloalkyl and R₅in R- or S-1-phenylethyl or a benzyl substituted in one or morepositions with a substituent selected from the group consisting of halo,amino, acetamido, C₁–C₁₀ haloalkyl and sulfo, wherein the sulfoderivative is a salt; R₂ is a C₂–C₁₀ alkyne of the formula R^(d)—C═C—where R^(d) is a C₁–C₈ alkyl; or R₂ is a halo, C₁–C₁₀ alkylamino, orC₁–C₁₀ alkylthio, R^(a) is hydrogen, R^(b) is C₁–C₁₀ alkyl and R₅ is asubstituted benzyl.
 35. A method according to claim 13, wherein saidactive ingredient is an A3 selective A3RAg that is selected from thegroup consisting of those of formula (IV) in which: X₁ isR^(a)R^(b)NC(═O), wherein R^(a) and R^(b) may be the same or differentand are selected from the group consisting of hydrogen, C₁–C₁₀ alkyl,amino, C₁–C₁₀ haloalkyl, C₁–C₁₀ aminoalkyl, and C₃–C₁₀ cycloalkyl, R₂ isselected from the group consisting of hydrogen, halo, C₁–C₁₀ alkyoxy,amino, C₂–C₁₀ alkenyl, and C₂–C₁₀ alkynyl, and R₄ is selected from thegroup consisting of R- and S-1-phenylethyl, an unsubstituted benzylgroup, and a benzyl group substituted in one or more positions with asubstituent selected from the group consisting of C₁–C₁₀ alkyl, amino,halo, C₁–C₁₀ haloalkyl, nitro, hydroxy, acetamido, C₁–C₁₀ alkoxy, andsulfo.
 36. A method according to claim 35, wherein said activeingredient is an A3 selective A3RAg that is selected from the groupconsisting of those of formula (IV) in which: R^(a) and R^(b) are thesame or different and are selected from the group consisting of hydrogenand C₁–C₁₀ alkyl, and R₂ is hydrogen or halo; R^(a) is hydrogen, R₂ ishydrogen and R₅ is unsubstituted benzyl; R^(b) is C₁–C₁₀ alkyl or C₃–C₁₀cycloalkyl and R₅ in R- or S-1-phenylethyl or a benzyl substituted inone or more positions with a substituent selected from the groupconsisting of halo, amino, acetamido, C₁–C₁₀ haloalkyl and sulfo,wherein the sulfo derivative is a salt; R₂ is a C₂–C₁₀ alkyne of theformula R^(d)—C═C— where R^(d) is a C₁–C₈ alkyl; or R₂ is a halo, C₁–C₁₀alkylamino, or C₁–C₁₀ alkylthio, R^(a) is hydrogen, R^(b) is C₁–C₁₀alkyl and R₅ is a substituted benzyl.
 37. A method for inhibitingabnormal cell proliferation in a subject in need thereof, comprisingadministering to the subject an adenosine A3 receptor agonist (A3RAg) inan amount of less than 100 μg/Kg body weight.
 38. A method according toclaim 37 wherein the amount of the A3RAg is less than 50 μg/kg bodyweight.